Rat Left Ventricular Myocytes Research Articles

Inhibiting glycosphingolipids alleviates cardiac hypertrophy by reducing reactive oxygen species and restoring autophagic homeostasis.

Cardiac hypertrophy is a compensatory stress response produced by a variety of factors, and pathologic hypertrophy can lead to irreversible, severe cardiac disease. Glycosphingolipids (GSLs) are vital constituents of cells, and changes in their content and composition are important factors causing mitochondrial dysfunction in diabetic cardiomyopathy; however, the relationship between GSLs expression and cardiac hypertrophy and specific mechanisms associated with it are not clear. Here, using male C57BL/6 mice, we performed aortic arch reduction surgery to establish an animal model of pressure overload cardiac hypertrophy. In addition, phenylephrine was used in vitro to induce H9c2 cells and neonatal rat left ventricular myocytes (NRVMs) to establish a cellular hypertrophy model. Mass spectrometry revealed that the composition of GSLs was altered in pressure overload-induced hypertrophied mouse hearts and in stimulated hypertrophied cardiomyocyte cell lines. Specifically, in both cases, the proportion of endogenous lactosylceramide (LacCer) was significantly higher than in controls. Inhibition of GSL synthesis with Genz-123346 in NRVMs reduced cell hypertrophy, as well as fibrosis and apoptosis. By Western blotting, we detected decreased intracellular expression of Sirt3 and elevated phosphorylation of JNK after phenylephrine stimulation, but this was reversed in cells pretreated with Genz-123346. Additionally, increased protein expression of FoxO3a and Parkin, along with a decreased LC3-II/I protein ratio in phenylephrine-stimulated cells (compared with unstimulated cells), indicated that the mitochondrial autophagy process was disrupted; again, pretreatment with Genz-123346 reversed that. Our results revealed that changes in GSLs in cardiomyocytes, especially an increase of LacCer, may be a factor causing cellular hypertrophy, which can be alleviated by inhibition of GSLs synthesis. A possible mechanism is that GSLs inhibition increases the expression of Sirt3 protein, scavenges intracellular reactive oxygen species, and restores mitochondrial autophagy homeostasis, thereby lessening cardiomyocyte hypertrophy. In all, these results provide a new perspective for developing drugs for cardiac hypertrophy.

Rapid Pacing Decreases L-type Ca2+ Current and Alters Cacna1c Isogene Expression in Primary Cultured Rat Left Ventricular Myocytes

The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaL directly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+ channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaL in different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaL density by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of total cacna1c and of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaL density by 30%, mildly slowed ICaL inactivation and shifted steady-state inactivation to more negative potentials. Total cacna1c mRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaL density and cacna1c mRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes.Graphical

Reversible and Irreversible Effects of Electroporation on Contractility and Calcium Homeostasis in Isolated Cardiac Ventricular Myocytes.

Irreversible electroporation is an energy form utilizing high-voltage pulsed electric field, leading to cellular homeostasis disruption and cell death. Recently, irreversible electroporation has shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca2+ homeostasis and recovery of cardiac myocytes. Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca2+ were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field. Electroporation led to an immediate increase in intracellular Ca2+ which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP (140 V, 100 µs) increased sarcomere shortening, Ca2+ transient amplitude, and diastolic Ca2+ level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca2+ transient amplitude decreased further below pre-EP level and diastolic Ca2+ level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses. Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca2+ homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca2+ and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.

Structural variability of dyads relates to calcium release in rat ventricular myocytes

Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into ‘compact’ or ‘loose’ types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into ‘early’ and ‘late’ components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.

Effects of Atorvastatin on Transient Sodium Currents in Rat Normal, Simulated Ischemia, and Reperfusion Ventricular Myocytes

Objectives: (1) To detect the whether the effects of simulated ischemia on I_Na of rat left ventricular myocytes in a time-dependent manner and the effects of atorvastatin on ischemia I_Na; (2) To investigate the effects of atorvastatin on I_Na of rat-simulated ischemia/reperfusion (I/R) ventricular cells. Materials and Methods: Ventricular cells were enzymatically isolated by Langendorff perfusion system. Whole-cell patch clamp was applied to detect I_Na level. Some elements of extracellular fluid were hanged to simulate the status of normal, I and R condition. Then the effects of atorvastatin on I_Na were observed. Results: (1) During simulated reperfusion, I_Na decreased and atorvastatin further suppressed the reduction degree. (2) At test potential –40 mV, no difference was detected among peak I_Na amplitude of ischemia for 20 min, reperfusion phase 3/5/7/9 min in continuous ischemia (I) group (p = 0.275). In I/R group, peak I_Na amplitude continuously decreased at 3 min (p = 0.005) and 9 min (p = 0.041). In atorvastatin intervention + I/R (Statin + I/R) group, peak I_Na amplitude at reperfusion 3 min decreased compared with ischemia phase (p = 0.000), while no significant difference was detected between 3 and 9 min (p = 0.858). The differences were significant at the same time point between groups. At reperfusion 3/5/7/9 min, peak I_Na of the I/R group was lower than the ischemia group (all p = 0.000), same as the Statin + I/R group (p = 0.000, p = 0.003, p = 0.006, and p = 0.001). Peak I_Na of the Statin + I/R group was higher than the I/R group at the same time point (p = 0.011, p = 0.033, p = 0.003, p = 0.003). There was no change in the I group during reperfusion phase (p > 0.05). In I/R group, V_1/2 (mV) shifted from –58.87 ± 3.36 to –54.33 ± 2.40, k (mV) shifted from 1.25 ± 0.59 to 1.91 ± 0.84 (p < 0.05). In the Statin + I/R group, V_1/2 (mV) increased from –57.80 ± 2.97 to –52.76 ± 3.14 (p < 0.01), no change was observed in k (p > 0.05). Conclusions: (1) In the status of reperfusion, I_Na decreased more than that in the status of ischemia. (2) Atorvastatin protected the cells from reduction of I_Na during long-time simulated (>15 min) I/R. (3) Overall, atorvastatin affected I_Na of the normal, simulated ischemic/reperfusion cell in rat left ventricle by blocking sodium channel ­directly.

NF-κB inhibitor on Toll-like receptor 4 signal-induced expression of angiotensinogen and AT1a receptor in neonatal rat left ventricular myocytes.

Effects of toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway on expression of angiotensinogen and AT1a receptor were investigated, to explore the role of TLR4/NF-κB signaling pathway in cardiovascular disease. Neonatal rat left ventricular myocytes (NRVMs) were cultured and cardiomyocytes were identified by immunocytochemical staining of sarcomeric α-actin. NRVMs were treated with lipopolysaccharide (LPS) at a dose of 10, 100 and 1,000 ng/ml, and RT-PCR was performed 24 h later to detect the expression of TLR4, angiotensinogen (ATG) and AT1a at mRNA level. NRVMs were cultured and pretreated with caffeic acid phenethylester (CAPE) for 30 min. Then NRVMs were stimulated with LPS (1,000 ng/ml) for 24 h. Nuclear translocation of NF-κB p65 was detected by immunocytochemistry. Expression of TLR4, angiotensinogen and AT1a receptor after CAPE stimulation was detected by RT-PCR. TLR4 mRNA was highly expressed in in vitro cultured NRVMs, and the expression level was significantly increased by LPS (10–1,000 ng/ml) stimulation in a dose-dependent manner (P<0.05). LPS stimulation also significantly increased the expression levels of angiotensinogen and AT1a receptor in a dose-dependent manner (P<0.05). NF-κB was activated and nuclear translocation of NF-κB p65 occurred after stimulation with LPS (1,000 ng/ml) for 24 h, while CAPE (20 µg/ml) inhibited the nuclear translocation of NF-κB p65 and inhibited LPS-induced expression of angiotensinogen and AT1a receptor. With LPS stimulation, TLR4 signaling positively regulates the expression of TLR4 and upregulates the expression of angiotensinogen and AT1a receptor in NRVMs. CAPE, an inhibitor of NF-κB, inhibited NF-κB p65 activation and inhibited the upregulation of TLR4, angiotensinogen and AT1a receptors induced by LPS. These results suggest that NF-κB plays a key regulatory role in the above-mentioned effects induced by LPS. Intervention with TLR4/NF-κB signaling may become a new target for prevention and treatment of cardiovascular diseases.

Liguzinediol Enhances the Inotropic Effect of Rat Hearts via Inhibition of Protein Phosphatase (PP1 and PP2A) Activities.

It has been demonstrated that liguzinediol (2,5-dihydroxymethyl-3,6-dimethylpyrazine, LZDO), a derivative of ligustrazine from Ligusticum wallichii Franch, exerts positive inotropy in isolated rat heart mediated by the sarcoplasmic reticulum Ca-ATPase (SERCA2a). Here, we further explore the underlying mechanism of the positive inotropic effect of LZDO in rat hearts. In vivo and ex vivo rat heart experiments, biochemistry, and Western blot techniques were used to analyze the rat heart contractility, and SERCA2a activity, phospholamban (PLB) phosphorylation, and protein phosphatase (PP1 and PP2A) activities in rat left ventricular myocytes, respectively. LZDO (20 mg/kg) significantly increased the inotropy of rat heart in vivo. In isolated rat heart experiments, LZDO (100 μM) restored the decreased inotropy induced by caffeine (0.5 mM); however, calyculin A (4 nM), an inhibitor of PP1 and PP2A, eliminated the inotropic effect of LZDO (100 μM). Moreover, LZDO (1, 10, and 100 μM) significantly enhanced SERCA2a activity and increased the levels of phosphorylated PLB on both serine-16 (Ser-16) and threonine-17 (Thr-17). In addition, LZDO (100 μM) significantly inhibited the activities of PP1 and PP2A. The positive inotropic effects of LZDO on in vivo and ex vivo rat hearts seem to be mediated through inhibition of PP1/PP2A, which may suppress dephosphorylated PLB and enhance SERCA2a activity. LZDO may prove effective in treating heart failure in clinical settings based on its unique biological mechanism.

Cardiac inotropy, lusitropy, and Ca2+ handling with major metabolic substrates in rat heart.

Fatty acid (FA)-dependent oxidation is the predominant process for energy supply in normal heart. Impaired FA metabolism and metabolic insufficiency underlie the failing of the myocardium. So far, FA metabolism in normal cardiac physiology and heart failure remains undetermined. Here, we evaluate the mechanisms of FA and major metabolic substrates (termed NF) on the contraction, relaxation, and Ca2+ handling in rat left ventricular (LV) myocytes. Our results showed that NF significantly increased myocyte contraction and facilitated relaxation. Moreover, NF increased the amplitudes of diastolic and systolic Ca2+ transients ([Ca2+]i), abbreviated time constant of [Ca2+]i decay (tau), and prolonged the peak duration of [Ca2+]i. Whole-cell patch-clamp experiments revealed that NF increased Ca2+ influx via L-type Ca2+ channels (LTCC, ICa-integral) and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca2+]i trajectory to the right and increased [Ca2+]i for 50 % of sarcomere relengthening (EC50), suggesting myofilament Ca2+ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca2+ sensitivity, abolished the NF-induced enhancement of [Ca2+]i amplitude and the tau of [Ca2+]i decay, indicating the association of myofilament Ca2+ desensitization with the changes in [Ca2+]i profile in NF. NF reduced intracellular pH ([pHi]). Increasing [pH]i buffer capacity with HCO3/CO2 attenuated Δ [pH]i and reversed myofilament Ca2+ desensitization and Ca2+ handling in NF. Collectively, greater Ca2+ influx through LTCCs and myofilament Ca2+ desensitization, via reducing [pH]i, are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-016-1892-8) contains supplementary material, which is available to authorized users.

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 μM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.

Arsenic trioxide and resveratrol show synergistic anti-leukemia activity and neutralized cardiotoxicity.

Cardiotoxicity is an aggravating side effect of many clinical antineoplastic agents such as arsenic trioxide (As2O3), which is the first-line treatment for acute promyelocytic leukemia (APL). Clinically, drug combination strategies are widely applied for complex disease management. Here, an optimized, cardiac-friendly therapeutic strategy for APL was investigated using a combination of As2O3 and genistein or resveratrol. Potential combinations were explored with respect to their effects on mitochondrial membrane potential, reactive oxygen species, superoxide dismutase activity, autophagy, and apoptosis in both NB4 cells and neonatal rat left ventricular myocytes. All experiments consistently suggested that 5 µM resveratrol remarkably alleviates As2O3-induced cardiotoxicity. To achieve an equivalent effect, a 10-fold dosage of genistein was required, thus highlighting the dose advantage of resveratrol, as poor bioavailability is a common concern for its clinical application. Co-administration of resveratrol substantially amplified the anticancer effect of As2O3 in NB4 cells. Furthermore, resveratrol exacerbated oxidative stress, mitochondrial damage, and apoptosis, thereby reflecting its full range of synergism with As2O3. Addition of 5 µM resveratrol to the single drug formula of As2O3 also further increased the expression of LC3, a marker of cellular autophagy activity, indicating an involvement of autophagy-mediated tumor cell death in the synergistic action. Our results suggest a possible application of an As2O3 and resveratrol combination to treat APL in order to achieve superior therapeutics effects and prevent cardiotoxicity.

Acute Administration of Chitosan Nanoparticles Increases Ca&lt;sup&gt;2+&lt;/sup&gt; Leak in Rat Cardiomyocytes

Polymeric nanoparticles like chitosan nanoparticles may be used to deliver drugs to particular organs, such as heart. However, due to the lack of information about acute effects of chitosan nanoparticles in cardiac calcium handling, we evaluated the same in intact rat left ventricular myocytes. Chitosan nanoparticles were synthesized by ionic gelation method for three different concentrations of chitosan and tripolyphosphate (TPP) such as 1:1, 2:1 and 3:1, respectively. The size of the particles was below 100 nm for the 2:1 and 3:1 chitosan:TPP ratio and 300 nm for 1:1 ratio. The particles synthesized in 3:1 ratio were incubated for 0, 15, 30 and 60 minutes with Fluo-3 loaded cardiomyocytes, their effects were evaluated in local Ca2+ release events using confocal microscopy and compared with control cells. Chitosan nanoparticles increased the amplitude and size of Ca2+ spark by 14.1% and 24.1% at 30 minutes of incubation; while the increment was 24.7% and 28.4% at 60 minutes respectively. Accordingly, rising time of Ca2+ sparks was decreased by 47% at 30 minutes. These changes were reflected in increased local Ca2+ flux by 58.3% and spark-mediated Ca2+ leak by 145.9% and 146.5% at 30, and 60 minutes, respectively. Hence, these results indicate that chitosan nanoparticles modify the properties of local Ca2+ release events mainly at short incubation times and must be taken into account while using these nanoparticles in drug delivery.

Localization of Kv4.2 and KChIP2 in lipid rafts and modulation of outward K+ currents by membrane cholesterol content in rat left ventricular myocytes.

Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K(+)-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K(+) current (I to), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K(+) outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K(+) currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I to (20.0 ± 1.6pApF(-1) vs. 28.5 ± 2.0pApF(-1), n = 15, p < 0.001, V Pip = 40 mV) within 5 min. Control solution resulted in a significantly smaller reduction in I to (17 ± 3 %, p < 0.001, p < 0.01 compared with MBCD). MBCD induced a 38 ± 9 % increase in the non-inactivating current component (I sus) (10.1 ± 0.6pApF(-1) vs. 7.6 ± 0.4pApF(-1), n = 15, p < 0.001). This effect was absent in control solution. The increase in I sus was not sensitive to 100 μM 4-aminopyridine or 20 mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 unlikely. In conclusion, in rat ventricular cardiomyocytes, a fraction of Kv4.2 and KChIP2 is localized in lipid rafts. Membrane cholesterol depletion results in ~12 % net reduction of I to, a redistribution of the channel proteins Kv4.2 and KChIP2 and an increased delayed rectifier current.

Lipid Rescue Reverses the Bupivacaine-induced Block of the Fast Na+ Current (INa) in Cardiomyocytes of the Rat Left Ventricle

Cardiovascular resuscitation upon intoxication with lipophilic ion channel-blocking agents has proven most difficult. Recently, favorable results have been reported when lipid rescue therapy is performed, i.e., the infusion of a triglyceride-rich lipid emulsion during resuscitation. However, the mechanism of action is poorly understood. The authors investigate the effects of a clinically used lipid emulsion (Lipovenös® MCT 20%; Fresenius Kabi AG, Bad Homburg, Germany) on the block of the fast Na current (INa) induced by the lipophilic local anesthetic bupivacaine in adult rat left ventricular myocytes by using the whole cell patch clamp technique. Bupivacaine at 10 µm decreased INa by 54% (-19.3 ± 1.9 pApF vs. -42.3 ± 4.3 pApF; n = 17; P < 0.001; VPip = -40 mV, 1 Hz). Addition of 10% lipid emulsion in the presence of bupivacaine produced a 37% increase in INa (-26.4 ± 2.8 pApF; n = 17; P < 0.001 vs. bupivacaine alone). To test whether these results could be explained by a reduction in the free bupivacaine concentration by the lipid (lipid-sink effect), the authors removed the lipid phase from the bupivacaine-lipid mixture by ultracentrifugation. Also, the resulting water phase led to an increase in INa (+19%; n = 17; P < 0.001 vs. bupivacaine), demonstrating that part of the bupivacaine had been removed during ultracentrifugation. The substantially less lipophilic mepivacaine (40 µm) reduced INa by 27% (n = 24; P < 0.001). The mepivacaine-lipid mixture caused a significant increase in INa (+17%; n = 24; P < 0.001). For mepivacaine, only a small lipid-sink effect could be demonstrated (+8%; n = 23; P < 0.01), reflecting its poor lipid solubility. The authors demonstrate lipid rescue on the single-cell level and provide evidence for a lipid-sink mechanism.

Xanthine oxidase inhibition preserves left ventricular systolic but not diastolic function in cardiac volume overload

Xanthine oxidase (XO) is increased in human and rat left ventricular (LV) myocytes with volume overload (VO) of mitral regurgitation and aortocaval fistula (ACF). In the setting of increased ATP demand, XO-mediated ROS can decrease mitochondrial respiration and contractile function. Thus, we tested the hypothesis that XO inhibition improves cardiomyocyte bioenergetics and LV function in chronic ACF in the rat. Sprague-Dawley rats were randomized to either sham or ACF ± allopurinol (100 mg·kg(-1)·day(-1), n ≥7 rats/group). Echocardiography at 8 wk demonstrated a similar 37% increase in LV end-diastolic dimension (P < 0.001), a twofold increase in LV end-diastolic pressure/wall stress (P < 0.05), and a twofold increase in lung weight (P < 0.05) in treated and untreated ACF groups versus the sham group. LV ejection fraction, velocity of circumferential shortening, maximal systolic elastance, and contractile efficiency were significantly depressed in ACF and significantly improved in ACF + allopurinol rats, all of which occurred in the absence of changes in the maximum O2 consumption rate measured in isolated cardiomyocytes using the extracellular flux analyzer. However, the improvement in contractile function is not paralleled by any attenuation in LV dilatation, LV end-diastolic pressure/wall stress, and lung weight. In conclusion, allopurinol improves LV contractile function and efficiency possibly by diminishing the known XO-mediated ROS effects on myofilament Ca(2+) sensitivity. However, LV remodeling and diastolic properties are not improved, which may explain the failure of XO inhibition to improve symptoms and hospitalizations in patients with severe heart failure.

Effect of trimetazidine treatment on the transient outward potassium current of the left ventricular myocytes of rats with streptozotocin-induced type 1 diabetes mellitus

Cardiovascular complications are a leading cause of mortality in patients with diabetes mellitus (DM). The present study was designed to investigate the effects of trimetazidine (TMZ), an anti-angina drug, on transient outward potassium current (Ito) remodeling in ventricular myocytes and the plasma contents of free fatty acid (FFA) and glucose in DM. Sprague-Dawley rats, 8 weeks old and weighing 200-250 g, were randomly divided into three groups of 20 animals each. The control group was injected with vehicle (1 mM citrate buffer), the DM group was injected with 65 mg/kg streptozotocin (STZ) for induction of type 1 DM, and the DM+TMZ group was injected with the same dose of STZ followed by a 4-week treatment with TMZ (60 mg·kg−1·day−1). All animals were then euthanized and their hearts excised and subjected to electrophysiological measurements or gene expression analyses. TMZ exposure significantly reversed the increased plasma FFA level in diabetic rats, but failed to change the plasma glucose level. The amplitude of Ito was significantly decreased in left ventricular myocytes from diabetic rats relative to control animals (6.25 ± 1.45 vs 20.72 ± 2.93 pA/pF at +40 mV). The DM-associated Ito reduction was attenuated by TMZ. Moreover, TMZ treatment reversed the increased expression of the channel-forming alpha subunit Kv1.4 and the decreased expression of Kv4.2 and Kv4.3 in diabetic rat hearts. These data demonstrate that TMZ can normalize, or partially normalize, the increased plasma FFA content, the reduced Ito of ventricular myocytes, and the altered expression Kv1.4, Kv4.2, and Kv4.3 in type 1 DM.

Effects of simulated ischemia-reperfusion and atorvastatin on INa in rat left ventricular myocytes.

ObjectiveTo observe time dependent effects of simulated ischemia-reperfusion on transient sodium currents (INa) in rat left ventricular myocytes, and effects of atorvastatin on INa in the condition of ischemia and...

E0232 The change of ventricular INa at different time of simulated ischaemia and the effect of atorvastatin

ObjectiveTo observe time dependent effects of simulated ischaemia on transient sodium currents (INa) of rat left ventricular myocytes, and the effects of atorvastatin on ischaemia INa.Methods30 Wistar rats were used...

Expression of angiotensinongen and AT1 receptor in cardiac myocytes after intervention of Toll-like receptor 4/NF-κB signaling

Objective To investigate the expression of angiotensinongen and AT1 receptor in cardiac myocytes after intervention of Toll-like receptor 4 (TLR4)/NF-κB signaling. Design and methods Cultured neonatal rat left ventricular myocytes (NRVMs) were divided into 3 groups randomly. ① Control: DMEM medium without any intervention; ② lipopolysaccharide (LPS 1000ng/ml) group: DMEM with LPS 1000ng/ml to activate TLR4/NF-κB signaling;③ LPS+Caffeic Acid Phenethyl Ester (CAPE) group: pretreated with CAPE (20μg/mL), NF-κB inhibitor, for 30min before LPS incubation.

Metabolites of MDMA Induce Oxidative Stress and Contractile Dysfunction in Adult Rat Left Ventricular Myocytes

Repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy) produces eccentric left ventricular (LV) dilation and diastolic dysfunction. While the mechanism(s) underlying this toxicity are unknown, oxidative stress plays an important role. MDMA is metabolized into redox cycling metabolites that produce superoxide. In this study, we demonstrated that metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes. Metabolites of MDMA used in this study included alpha-methyl dopamine, N-methyl alpha-methyl dopamine and 2,5-bis(glutathion-S-yl)-alpha-MeDA. Dihydroethidium was used to detect drug-induced increases in reactive oxygen species (ROS) production in ventricular myocytes. Contractile function and changes in intracellular calcium transients were measured in paced (1 Hz), Fura-2 AM loaded, myocytes using the IonOptix system. Production of ROS in ventricular myocytes treated with MDMA was not different from control. In contrast, all three metabolites of MDMA exhibited time- and concentration-dependent increases in ROS that were prevented by N-acetyl-cysteine (NAC). The metabolites of MDMA, but not MDMA alone, significantly decreased contractility and impaired relaxation in myocytes stimulated at 1 Hz. These effects were prevented by NAC. Together, these data suggest that MDMA-induced oxidative stress in the left ventricle can be due, at least in part, to the metabolism of MDMA to redox active metabolites.

Length-Tension Relationships Are Similar Between Cardiac Myocytes And Skeletal Muscle Fibers

According to the Frank-Starling relationship increased ventricular volume increases ventricular output, which helps match peripheral energy supply with demand. The cellular basis for this relationship is, in large part, the myofilament length-tension relationship. The length-tension relationship in maximally activated skinned preparations is relatively shallow and similar between cardiac myocytes and skeletal muscle fibers. During twitch activations the length tension relationship becomes steeper in both cardiac and skeletal muscle; however, it remains unclear whether length dependence of tension differs between striated muscle cell types during submaximal activations. The purpose of this study was to compare myofilament length-tension relationships between cardiac myocytes, fast-twitch and slow twitch skeletal muscle fibers during half-maximal calcium activations. For these experiments, passive and active sarcomere length-tension relationships were characterized in skinned rat left ventricular cardiac myocytes, fast-twitch and slow-twitch skeletal muscle fibers. For active length-tension relationships, cells were mounted between a force transducer and motor and activated to yield 50-65% maximal force after which isometric force was measured over a range of ascending limb sarcomere lengths monitored by IonOptix SarLen system. Passive tension was considerably greater in cardiac myocytes (3.5 ± 2.5 kN/m2 at 2.30 ± 0.04μm) compared to both fast-twitch (1.1 ± 0.8 kN/m2 at 2.70 ± 0.07μm) and slow-twitch (1.4 ± 0.5 kN/m2 at 2.70 ± 0.07μm) muscle fibers. Active myofilament length-tension relationships were considerably shallower in slow-twitch fibers compared to fast-twitch fibers. Interestingly, cardiac myocytes exhibited two distinct populations of length-tension relationships, one nearly identical to fast-twitch fibers and the other similar to slow-twitch fibers. These results indicate that cardiac myocytes exhibit length-tension relationships very similar to skeletal muscle fibers. The finding of two populations of cardiac myocyte length-tension relationships suggests a mode for adjusting Frank-Starling relationships following acute and/or chronic ventricular adaptations.