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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

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 (IKs ) to the aging-related remodeling of the heart as well as the role of insulin treatment on IKs 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 IKs . 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 IKs in β3 -ARs-stimulated cells could be reversed with a PKG inhibitor, indicating the correlation between PKG-activation and β3 -ARs activation. Furthermore, in vivo treatment of aged rats, characterized by β3 -ARs activation, with either insulin or a PKG inhibitor for 2 weeks provided significant recoveries in IKs , 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 β3 -ARs overexpressed cells. The inhibition of IKs in aged-rat cardiomyocytes seems to be associated with activated β3 -ARs dependent remodeling in the interaction between KCNQ1 and KCNE1. Significant recoveries in ventricular-repolarization of insulin-treated aged cardiomyocytes via recovery in IKs strongly emphasize two important issues: (1) IKs 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 IKs via affecting the β3 -ARs signaling pathway and particularly affecting activated PKG.

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 Zn2+ ([Zn2+](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 [Zn2+](i), which can further precipitate in cleavage of extracellular matrix in the heart.

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+/Ca2+-exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca2+ level and depressed SR Ca2+ 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 Ca2+ 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.

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.

Calmodulin (CaM) mutants are associated with severe arrhythmias (calmodulinopathy). While dysregulation of Ca 2+ handling proteins (RyR2, Ca V 1.2) are known mediators of calmodulinopathy, the role of Na + channels (Na V s) remains unclear. Interestingly, effects on Ca V 1.2 do not fully explain action potential (AP) prolongation by the long QT syndrome CaM mutant D96V. We investigated Na V dysregulation by D96V-CaM and its arrhythmogenic impact. In wild-type (WT) murine cardiomyocytes (CM) dialysis of D96V-CaM (6.5 μM) via a patch pipette prolonged AP, induced early and delayed afterdepolarizations (EADs and DADs) and increased Ca 2+ waves compared to WT-CaM. The effects of D96V-CaM were abrogated by a Na V 1.6 blocker, 4,9-anhydrotetrodotoxin (4,9ahTTX, 300 nM; Fig. 1A ). Dialysis of D96V-CaM dysregulated inactivation of 4,9ahTTX-sensitive Na + current (I Na ) promoting late I Na in WT murine CM ( Fig. 1B ) and in Chinese Hamster Ovary (CHO) cells expressing Na V 1.6 but not Na V 1.5. D96V-CaM, but not WT-CaM, also promoted late I Na in human induced pluripotent stem cell (iPSC) - derived ventricular myocytes, underscoring translational relevance ( Fig. 1C ). To examine the arrhythmia potential of Na V 1.6 dysregulation by D96V-CaM, we generated mice with cardiac-specific expression of D96V-CaM (cD96V; tgD96V-CaM x β-MHC-Cre). cD96V mice exhibited increased late I Na and Ca 2+ waves resembling those in D96V-CaM-dialyzed WT CMs. In vivo cD96V mice evidenced prolonged QTc intervals and ventricular tachycardia (VT) during bradycardia (carbachol 0.5 mg/kg intraperitoneal). Cardiac-specific Na V 1.6 knockout protected cD96V mice from impaired I Na inactivation and arrhythmogenic Ca 2+ waves on cellular level as well as long QT and VT in vivo ( Fig. 1D, E ). In summary, D96V-CaM promotes arrhythmias by dysregulating Na V 1.6 and thereby, facilitating aberrant Ca 2+ release. Importantly, Na V 1.6 may be a therapeutic target for management of CaM-related arrhythmias.

Calmodulin facilitates normal sodium channel (Na V ) inactivation, necessary to prevent proarrhythmic late sodium current (I Na ). Previous studies have implicated Na V 1.6 dysfunction in late I Na -mediated arrhythmias. Therefore we investigated Na V dysregulation by arrhythmogenic CaM mutant D96V. Chinese hamster ovary cells expressing Na V 1.6 were transfected with either WT- or D96V-CaM. STED microscopy revealed enlarged Na V 1.6 clusters in cells receiving D96V-CaM relative to WT. Therefore we explored Na V 1.6 remodeling in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V). Confocal microscopy confirmed expression of Na V 1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RyR2 in cD96V hearts, consistent with T-tubular localization. STORM microscopy enabled the quantitative assessment of Na V 1.6 clustering. In both WT and cD96V hearts, ~50% of Na V 1.6 clusters localized V 1.6 density within these regions increased 67% in cD96V relative to WT. We probed the functional impact of this surprising remodeling using SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings. The number of NaVs as well as frequency of late Na V 1.6 burst openings were significantly increased in cD96V myocytes relative to WT (Fig 1). Previous work implicates aberrant late Na V 1.6 activity in proarrhythmic Ca2+ mishandling. Indeed, STORM revealed significant enrichment of NCX near Na V 1.6 in both WT and cD96V. Consistent with these data cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. This effect was reversed by cardiac-specific Na V 1.6 knockout. To our knowledge, this is the first report of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, which may shed mechanistic insight into calmodulinopathy.

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized

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

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-mu M 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-mu M 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.

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 Zn2+ level ([Zn2+]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 Zn2+-transporters responsible for [Zn2+]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 Zn2+-transporters, responsible for Zn2+-influx into cytosol, ZIP7 and ZIP14 were increased with significant decrease in ZIP8 of MetS-rat cardiomyoctes, while Zn2+-transporters, responsible for cytosolic Zn2+-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 Zn2+-regulation via Zn2+-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.

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 (IK) were recorded in the presence of external Cd2+ and both inward and outward parts of the current were calculated, while their expression levels were determined with qPCR. The levels of intracellular free Ca2+ and H+ (pHi) 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 IK in a concentration-dependent manner. Sibutramine (10 μM) induced Ca2+-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. pHi inhibition by sibutramine supports the observed negative alterations in contractility. Changes in mRNA levels of different IK subunits are consistent with the acute inhibition of the repolarizing IK, affecting AP parameters, and provoke the cardiotoxicity.

The balanced presence of polyunsaturated fatty acids and specific bioactive components of high-polyphenol extra virgin olive oil (HP-EVOO)result in the presumed health benefit. Enrichment of its principal polyphenols is shown to be protective in oxidative damage and improves mitochondrial dysfunction. We investigated the effect of different concentrations of HP-EVOO applications on mitochondrial functions and antioxidant defense status on human keratinocyte cells (HaCaT) under the H2O2 induced stress conditions. Our data showed that HP-EVOO has prominentantioxidant capacityin 2%, 5% and 10% HP-EVOO groups (p = 0.000) especially under H2O2 toxicity. Mitochondrial gradients for ATP production were preserved with the increase in mitochondrial membrane potential (MMP) with 2% (p = 0.001), 5% (p = 0.000), 10% (p = 0.003) EVOO application, which resulted in improving cell viability.Hereby, it was concluded that EVOO might have a beneficial effect on skin health by increasing antioxidant status and by providing higher MMP to maintain the mitochondrial function of the keratinocytes in our study.

Role of β3 -AR dysregulation, as either cardio-conserving or cardio-disrupting mediator, remains unknown yet. Therefore, we examined the molecular mechanism of β3 -AR activation in depressed myocardial contractility using a specific agonist CL316243 or using β3 -AR overexpressed cardiomyocytes. Since it has been previously shown a possible correlation between increased cellular free Zn2+ ([Zn2+ ]i ) and depressed cardiac contractility, we first demonstrated a relation between β3 -AR activation and increased [Zn2+ ]i , parallel to the significant depolarization in mitochondrial membrane potential in rat ventricular cardiomyocytes. Furthermore, the increased [Zn2+ ]i induced a significant increase in messenger RNA (mRNA) level of β3 -AR in cardiomyocytes. Either β3 -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 β3 -AR activation induced depression in both Na+ - and Ca2+ -currents, the prolonged action potential (AP) seems to be associated with a marked depression in K+ -currents. The β3 -AR activation caused a negative inotropic effect on the mechanical activity of the heart, through affecting the cellular Ca2+ -handling, including its effect on Ca2+ -leakage from sarcoplasmic reticulum (SR). Our cellular level data with β3 -AR agonism were supported with the data on high [Zn2+ ]i and β3 -AR protein-level in metabolic syndrome (MetS)-rat heart. Overall, our present data can emphasize the important deleterious effect of β3 -AR activation in cardiac remodeling under pathological condition, at least, through a cross-link between β3 -AR activation, NO-signaling, and [Zn2+ ]i pathways. Moreover, it is interesting to note that the recovery in ER-stress markers with β3 -AR agonism in hyperglycemic cardiomyocytes is favored. Therefore, how long and to which level the β3 -AR agonism would be friend or become foe remains to be mystery, yet.

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 Ca2+-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 Ca2+-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 Ca2+-handling, implying its possible protective role of mitochondria-targeting antioxidant-treatment during aging.

Objectives:It has been reported that sodium glucose co-transporter (SGLT) inhibitors lowering blood glucose in diabetic patients via inhibiting glucose re-absorption. Although it has not been clearly identified the effects of these inhibitors on heart function, these inhibitors may act like Na+/H+-exchanger (NHE) inhibitors. In this study, we were aimed to clarify the underlying cellular mechanism of a SGLT2-inhibitor, Dapagliflozin (DAPA), on hyperglycemic embryonic rat ventricular cells (H9c2 cell line) via electrophysiological measurements.Materials and Methods:One group of H9c2 cells were incubated with high glucose (25 mM) medium for 24-hours and 48-hours at 37 °C to obtain hyperglycemic (HG) cardiomyocytes. Another group of H9c2 cells were incubated together with high glucose medium and DAPA (D185360, TORONTO Research Chemicals; 100 nM or 1 μM). Intracellular ion concentrations, ([X]in), reactive oxygen species, ([ROS]in) and mitochondrial membrane potential, (MMP) were monitored via specific fluorescent dyes (DCFDA, FluoZin-3AM, JC-1, SNARF, FURA-2AM and SBFI) with confocal microscope and microspectrofluorometer. All data are presented as mean (± SEM) and statistical analysis performed by student t-test. Significance level considered at p

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.

Intracellular labile (free) Zn2+-level ([Zn2+](i)) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn2+](i) is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn2+](i) on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn2+](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 [Zn2+](i) increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn2+](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-[Zn2+](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-[Zn2+](i) with no change in mRNA level of Kv4.2, while the mRNA level of I-KATP subunit, SUR2A was increased significantly with increased-[Zn2+](i), being reversible with DTT. Overall we demonstrated that high-[Zn2+](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 [Zn2+](i) can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress.

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 (5 mg/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.

Rosmarinic acid (RA), which has multiple bioactive properties, might be a useful agent for protecting central nervous system against age related alterations. In this context, the purpose of the present study was to investigate possible protective effects of RA on mismatch negativity (MMN) component of auditory event-related potentials (AERPs) as an indicator of auditory discrimination and echoic memory in the ovariectomized (OVX) rats injected with D-galactose combined with neurochemical and histological analyses. Ninety female Wistar rats were randomly divided into six groups: sham control (5); RA-treated (R); OVX (O); OVX + RA-treated (OR); OVX+ D-galactose-treated (OD); OVX+ D-galactose + RA treated (ODR). Eight weeks later, MMN responses were recorded using the oddball condition. An amplitude reduction of some components of AERPs was observed due to ovariectomy with or without D-galactose administiration and these reduction patterns were diverse for different electrode locations. MMN amplitudes were significantly lower over temporal and right frontal locations in the o and OD groups versus the S and R groups, which was accompanied by increased thiobarbituric acid reactive substances (TBARS) and hydroxy-2-nonenal (4-HNE) levels. RA treatment significantly increased AERP/MMN amplitudes and lowered the TBARS/4-HNE levels in the OR and ODR groups versus the 0 and OD groups, respectively. Our findings support the potential benefit of RA in the prevention of auditory distortion related to the estrogen deficiency and D-galactose administration at least partly by antioxidant actions. (C) 2017 Elsevier B.V. All rights reserved.

Background: The objective of this study was to examine the effect of swimming exercise on aging-related Ca 2+ 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 Ca 2+ currents and associated Ca 2+ 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, Ca 2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca 2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca 2+ 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 Ca 2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

Ticagrelor, a P2Y12-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-mu M palmitic acid for 24-h) H9c2 cardiomyocytes in the presence or absence of ticagrelor (1-mu M 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 Ca2+ and mRNA level of P2Y12 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.

Background Atrial fibrillation (AF) is a comorbidity associated with heart failure and catecholaminergic polymorphic ventricular tachycardia. Despite the Ca 2+ ‐dependent nature of both of these pathologies, AF often responds to Na + channel blockers. We investigated how targeting interdependent Na + /Ca 2+ dysregulation might prevent focal activity and control AF. Methods and Results We studied AF in 2 models of Ca 2+ ‐dependent disorders, a murine model of catecholaminergic polymorphic ventricular tachycardia and a canine model of chronic tachypacing‐induced heart failure. Imaging studies revealed close association of neuronal‐type Na + channels (nNa v ) with ryanodine receptors and Na + /Ca 2+ exchanger. Catecholamine stimulation induced cellular and in vivo atrial arrhythmias in wild‐type mice only during pharmacological augmentation of nNa v activity. In contrast, catecholamine stimulation alone was sufficient to elicit atrial arrhythmias in catecholaminergic polymorphic ventricular tachycardia mice and failing canine atria. Importantly, these were abolished by acute nNa v inhibition (tetrodotoxin or riluzole) implicating Na + /Ca 2+ dysregulation in AF. These findings were then tested in 2 nonrandomized retrospective cohorts: an amyotrophic lateral sclerosis clinic and an academic medical center. Riluzole‐treated patients adjusted for baseline characteristics evidenced significantly lower incidence of arrhythmias including new‐onset AF, supporting the preclinical results. Conclusions These data suggest that nNa V s mediate Na + ‐Ca 2+ crosstalk within nanodomains containing Ca 2+ release machinery and, thereby, contribute to AF triggers. Disruption of this mechanism by nNa v inhibition can effectively prevent AF arising from diverse causes.

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 Ca2+‐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 Ca2+ ([Ca2+]i) overload, indicative of disrupted cellular Ca2+‐homeostasis. Interestingly, increased [Ca2+]i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na+/Ca2+‐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 [Ca2+]i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca2+‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca2+]i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca2+]i and cardiac function with advanced age.

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 oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from fatty acid-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.

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.

The Zn2+ 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 ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ 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 ZnPO4- 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 Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-mu M ZnPT minute applications by using confocal microscopy. We also showed that 1-mu M ZnPT incubation induced significant increases in the phosphorylation levels of GSK3 beta (Ser21 and Ser9), Akt (Ser473), and NF kappa B (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 Zn2+ 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.

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 Zn2+ ([Zn2+]i) in cardiomyocytes under pathological conditions as well as the contribution of some re-expressed levels of Zn2+-transporters for redistribution of [Zn2+]i among suborganelles. Therefore, we first examined the cellular (total) [Zn2+] and then determined the protein expression levels of Zn2+-transporters in freshly isolated ventricular cardiomyocytes from 24-month rat heart compared to those of 6-month rats. The [Zn2+]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 [Zn2+]i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria ([Zn2+]SER and [Zn2+]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 [Zn2+]Mit with decreases in [Zn2+]SER. In addition, the re-distribution of these Zn2+-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 &micro, M, 5-h incubation), we provided an important data associated with the role of mitochondrial-ROS production in the [Zn2+]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 [Zn2+] distribution among cytosol and suborganelles with altered expression levels of the Zn2+-transporters.

Zinc is an important micronutrient for mammalians, whereas free/labile zinc ion (Zn2+) level ([Zn2+]i) in cells can be detrimental if it is over the physiological range. The cellular [Zn2+]i is regulated by proteins, responsible for its influx (ZIP family) into cells or efflux (ZnT-family) from cells. In addition, it has been shown that there is a close relationship between cellular [Zn2+]i and increasing oxidative stress, and both are also responsible for the dysregulation of the excitation/contraction coupling in the heart. Although age-dependent changes in the structure and function of the left ventricle are closely related to fibrosis, cellular evidence showed the importance of mitochondria as a potential target for aging medicine. However, several studies have shown that zinc supplementation provides important improvements in the maintenance of cardiovascular disorders, and it is needed to know the exact role of [Zn2+]i and its transporters in aging heart function. Although there is very little information related to the role of Zn2+ transporters in mammalian aging, some studies have shown their importance in cardiovascular disorders including failing human heart. Since few studies examined the role of ZIP14 as an inflammation responsive transporter and amplified with aging, we demonstrated an important data on the ZIP14 status in organelles such as the sarcoplasmic reticulum and mitochondria in aging rat’s ventricular cardiomyocytes. As a summary, we discussed our data in the light of literature data as well.

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 Zn2+ level in cardiomyocytes ([Zn2+](Mit)) is lower comparison to either its cytosolic or S(E)R level under physiological condition. However, there is little information about distribution of Zn2+-transporters on mitochondria and role of Zn2+-dependent mitochondrial-function associated with [Zn2+](Mit). Since we recently have shown how hyperglycemia (HG)-induced changes in ZIP7 and ZnT7 contribute to Zn2+-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 Zn2+-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 [Zn2+](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, OPAl, 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 Zn2+-muffling between cellular-compartments in HG or hypertrophic cardiomyocytes via affecting S(E)R-mitochondria coupling. Any alteration in this axis and/or cellular [Zn2+) may provide new insight for prevention/therapy of HF in diabetes and/or hypertrophy.

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