Search

Using produced polyclonal antibodies specific to the N-terminal sequence (residues 61-298) of rat obscurin, we investigated the isoform composition of this protein in 4 striated muscles: myocardium of the left ventricle, diaphragm, skeletal m. gastrocnemius (containing mainly fast fibers), and m. soleus (containing mainly slow fibers). The m. gastrocnemius, m. soleus, and diaphragm were found to have 2 giant isoforms of obscurin: a smaller A-isoform and a larger B-isoform. Their molecular weights were ~870 and ~1150 kDa in the diaphragm and m. gastrocnemius and ~880 and ~1130 kDa in m. soleus, respectively. The B-isoform to A-isoform ratio was 1:3 in the diaphragm and m. soleus and 1:4 in the m. gastrocnemius. In the left-ventricular myocardium, A-isoform of obscurin with a molecular weight of ~880 kDa was found. No other obscurin isoforms or their fragments within the molecular weight range of 10 up to ~800 kDa were revealed in the investigated rat striated muscles. The antibodies produced are recommended for research into qualitative and quantitative changes of giant obscurin isoforms in rat striated muscles in the norm and during the development of pathological processes., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca 2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca 2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca 2+ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca 2+ transients and sarcoplasmic reticulum (SR) Ca 2+ load, with a limited effect on L-type Ca 2+ channel-mediated Ca 2+ transients and SERCA-mediated Ca 2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca 2+ extrusion by Na + /Ca 2+ exchanger (NCX). MGO also increased the frequency of Ca 2+ waves during rest and these Ca 2+ release events were abolished by an external solution with zero [Na + ] and [Ca 2+ ]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca 2+ transients and SR Ca 2+ load, but it also triggered spontaneous Ca 2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca 2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na + ] revealed that MGO increases cytosolic [Na + ] by 57% from the maximal effect produced by the Na + -K + ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na + ] was a result of activation of a tetrodotoxin-sensitive Na + influx, but not an inhibition of Na + -K + ATPase. An increase in cytosolic [Na + ] after treating cells with ouabain produced similar effects on Ca 2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca 2+ regulation by increasing cytosolic [Na + ] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca 2+ extrusion by NCX, causing SR Ca 2+ overload and spontaneous Ca 2+ waves., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cardiovascular diseases are the leading cause of death in the world, cell therapies have been shown to recover cardiac function in animal models. Biomaterials used as scaffolds can solve some of the problems that cell therapies currently have, plasma polymerized pyrrole (PPPy) is a biomaterial that has been shown to promote cell adhesion and survival. The present research aimed to study PPPy nanoparticles (PPPyN) interaction with adult rat ventricular cardiomyocytes (ARVC), to explore whether PPPyN could be employed as a nanoscaffold and develop cardiac microtissues. PPPyN with a mean diameter of 330 nm were obtained, the infrared spectrum showed that some pyrrole rings are fragmented and that some fragments of the ring can be dehydrogenated during plasma synthesis, it also showed the presence of amino groups in the structure of PPPyN. PPPyN had a significant impact on the ARVC´s shape, delaying dedifferentiation, necrosis, and apoptosis processes, moreover, the cardiomyocytes formed cell aggregates up to 1.12 mm 2 with some aligned cardiomyocytes and generated fibers on its surface similar to cardiac extracellular matrix. PPPyN served as a scaffold for adult ARVC. Our results indicate that PPPyN-scaffold is a biomaterial that could have potential application in cardiac cell therapy (CCT)., (© 2021. The Author(s).)

The fast hybrid operator splitting (HOS) and stable uniformization (UNI) methods have been proposed to save computation cost and enhance stability for Markov chain model in cardiac cell simulations. Moreover, Chen-Chen-Luo's quadratic adaptive algorithm (CCL) combined with HOS or UNI was used to improve the tradeoff between speedup and stability, but without considering accuracy. To compromise among stability, acceleration, and accuracy, we propose a generalized Trotter operator splitting (GTOS) method combined with CCL independent of the asymptotic property of a particular ion-channel model. Due to the accuracy underestimation of the mixed root mean square error (MRMSE) method, threshold root mean square error (TRMSE) is proposed to evaluate computation accuracy. With the fixed time-step RK4 as a reference, the second-order GTOS combined with CCL (30.8-fold speedup) for the wild-type Markov chain model with nine states (WT-9 model) or (7.4-fold) for the wild-type Markov chain model with eight states (WT-8 model) is faster than UNI combined with CCL (15.6-fold) for WT-9 model or (1.2-fold) for WT-8 model, separately. Besides, the second-order GTOS combined with CCL has 3.81% TRMSE for WT-9 model or 4.32% TRMSE for WT-8 model more accurate than 72.43% TRMSE for WT-9 model or 136.17% TRMSE for WT-8 model of HOS combined with CCL. To compromise speedup and accuracy, low-order GTOS combined with CCL is suggested to have the advantages of high precision and low computation cost. For high-accuracy requirements, high-order GTOS combined with CCL is recommended. Graphical abstract.

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na + /HCO 3 - cotransporter (NBC) promotes the cellular co-influx of HCO 3 - and Na + . Since sAC activity is regulated by HCO 3 - , our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca 2+ transient (Ca 2+ i ) by epifluorescence, Ca 2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pH i ) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 μM), the selective inhibitor of sAC KH7 (1 μM), and the PKA inhibitor H89 (0.1 μM) induced a negative inotropic effect which was associated with a decrease in Ca 2+ i . Since PKA increases Ca 2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO 3 - influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca 2+ handling and basal cardiac contractility.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Ca v 1.2 L-type Ca 2+ channels and downstream Ca 2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Ca v 1.2 channels. This gives rise to a diminished Ca 2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca 2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca 2+ cycling and contractility. Whole cell currents through L-type Ca 2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Ca v 1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Ca v 1.2 channel function. We conclude that-against the currently prevailing view-basal Ca v 1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca 2+ cycling and contractility occurs independently of direct regulation of Ca v 1.2 channels by NO.

The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd 3+ (a non-specific blocker of mechanically gated current I MGCh , a component of late current I L ) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (I CaL ) nifedipine (10 μM) or verapamil (1 μM). In K + in /K + out or Cs + in /Cs + out media, blockade of I CaL produced no effect on I L at negative potentials, but inhibited I L at positive ones. In K + in /K + out medium, Gd 3+ (5 μM) decreased the net persistent current (I np ) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd 3+ alone or in combination with I CaL blockers shifted the reversal potential of I L to more negative values. At negative potentials, Gd 3+ decreased I K1 and inward current including I MGCh . At positive potentials, Gd 3+ alone or in combination with I CaL blockers decreased I L . When applied for 15 min in Cs + in /Cs + out medium at -45 mV, Gd 3+ produced no effect on net current and inward and outward components of I L . Thus, Gd 3+ can be viewed as a specific blocker of I MGCh only in Cs + medium.

Effects of ibutilide, a class III antiarrhythmic drug, on delayed rectifier potassium currents (I K ) in freshly isolated guinea pig ventricular myocytes were studied. Experiments were performed using the whole-cell configuration of patch-clamp technique under blockade of L-type calcium currents (Cav1). Ibutilide at concentrations ranging between 10 nM and 100 µM inhibited I Kr in dose-dependent manner with a half maximal effective concentration of 2.03 ± 0.74 µM (n = 5-10). The amplitude of tail currents activated by prepulse to + 20 mV was decreased from 253 ± 52 to 130 ± 25 pA (n = 8, p < 0.01) in the presence of 1 µM ibutilide. The envelope test revealed time-dependent changes in ratio of I K-tail /ΔI K during 0.2-2 s pulse durations in the absence of drug. With ibutilide, regardless of pulse duration, a relatively constant ratio was estimated, indicative of predominant involvement of I Kr component. The slow I Ks persisted to greater extent even at 100 μM ibutilide revealing a distinguishable selectivity toward the I Kr component.

Atrial natriuretic factor and brain natriuretic peptide are two important biomarkers in clinical cardiology. These two natriuretic peptide hormones are encoded by the paralogous genes Nppa and Nppb, which are evolutionary conserved. Both genes are predominantly expressed by the heart muscle during the embryonic and fetal stages, and in particular Nppa expression is strongly reduced in the ventricles after birth. Upon cardiac stress, Nppa and Nppb are strongly upregulated in the ventricular myocardium. Much is known about the molecular and physiological ques inducing Nppa and Nppb expression; however, the transcriptional regulatory mechanisms of the Nppa-Nppb cluster in vivo has proven to be quite complex and is not well understood. In this review, we will provide recent insights into the dynamic and complex regulation of Nppa and Nppb during heart development and hypertrophy, and the association of this gene cluster with the cardiomyocyte-intrinsic program of heart regeneration.

Inward rectifier potassium currents (I Kir,x ) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each I Kir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within I Kir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.

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 Ca 2+ 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 Ca 2+ transients ([Ca 2+ ] i ), abbreviated time constant of [Ca 2+ ] i decay (tau), and prolonged the peak duration of [Ca 2+ ] i . Whole-cell patch-clamp experiments revealed that NF increased Ca 2+ influx via L-type Ca 2+ channels (LTCC, I Ca-integral ) and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca 2+ ] i trajectory to the right and increased [Ca 2+ ] i for 50 % of sarcomere relengthening (EC 50 ), suggesting myofilament Ca 2+ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca 2+ sensitivity, abolished the NF-induced enhancement of [Ca 2+ ] i amplitude and the tau of [Ca 2+ ] i decay, indicating the association of myofilament Ca 2+ desensitization with the changes in [Ca 2+ ] i profile in NF. NF reduced intracellular pH ([pH i ]). Increasing [pH] i buffer capacity with HCO 3 /CO 2 attenuated Δ [pH] i and reversed myofilament Ca 2+ desensitization and Ca 2+ handling in NF. Collectively, greater Ca 2+ influx through LTCCs and myofilament Ca 2+ desensitization, via reducing [pH] i , are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF., Competing Interests: Compliance with ethical standards Conflicts of interests The authors declare that they have no conflicts of interest. Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2013068067); by the Brain Korea 21 Graduate Program of the Korean Ministry of Education, Science, and Technology, Seoul National University Hospital; the Korean Society of Hypertension (2013); SK Telecom Research Fund (no. 3420130290); and from the National Natural Science Foundation of China (NSFC, 31460265).

We studied the differences in twitch force of subendocardial and subepicardial cardiomyocytes isolated from mouse left ventricular wall at different preloads using an original single cell stretch method recently developed by us. Then, we used our mathematical models of subendocardial and subepicardial cells to predict underlying cellular mechanisms. Transmural differences in the amplitudes of active tension of subendocardial and subepicardial cardiomyocytes were revealed that could be related to the differences in cooperative end-to-end interaction between the neighboring regulatory units of the thin filament.

Alcohol consumption may result in electrocardiographic changes and arrhythmias, at least partly due to effects of ethanol on cardiac ionic currents. Contractility and intracellular Ca(2+) dynamics seem to be altered as well. In this study, we integrated the available (mostly animal) experimental data into previously published models of the rat and human ventricular myocytes to assess the share of ionic current components in ethanol-induced changes in AP configuration and cytosolic Ca(2+) transient in ventricular cardiomyocytes. The rat model reproduced well the experimentally observed changes in AP duration (APD) under ethanol (slight prolongation at 0.8 mM and shortening at ≥8 mM). These changes were almost exclusively caused by the ethanol-induced alterations of I K1. The cytosolic Ca(2+) transient decreased gradually with the increasing ethanol concentration as a result of the ethanol-induced inhibition of I Ca. In the human model, ethanol produced a dose-dependent APD lengthening, dominated by ethanol effect on I Kr, the key repolarising current in human ventricles. This effect might contribute to the clinically observed proarrhythmic effects of ethanol in predisposed individuals.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 μM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 μM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 μM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

In isolated human atrial cardiomyocytes, inhibition of K2P3.1 K(+) channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K2P3.1 (KCNK3), together with K2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K2P3.1 current is augmented, K2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (-6.7 ± 1.2 pA pF(-1) at -120 mV) strongly rectifying and Ba(2+)-sensitive (IC50 = 3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5%) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0%). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba(2+) block, drKir2.4 was the most sensitive (IC50 = 1.8 μM) and drKir2.1a the least sensitive channel (IC50 = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba(2+)-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances.

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5'-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of β1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy.

Echinochrome A (Ech A), a marine bio-product isolated from sea urchin eggs, is known to have cardioprotective effects through its strong antioxidant and ATP-sparing capabilities. However, the effects of Ech A on cardiac excitation-contraction (E-C) are not known. In this study, we investigated the effects of Ech A on cardiac contractility and Ca(2+) handling in the rat heart. In ex vivo Langendorff hearts, Ech A (3 μM) decreased left ventricular developing pressure to 77.7 ± 6.5 % of basal level. In isolated ventricular myocytes, Ech A reduced the fractional cell shortening from 3.4 % at baseline to 2.1 %. Ech A increased both diastolic and peak systolic intracellular Ca(2+) ([Ca(2+)]i). However, the ratio of peak [Ca]i to resting [Ca]i was significantly decreased. Ech A did not affect the L-type Ca(2+) current. Inhibiting the Na(+)/Ca(2+) exchanger with either NiCl2 or SEA400 did not affect the Ech A-dependent changes in Ca(2+) handling. Our data demonstrate that treatment with Ech A results in a significant reduction in the phosphorylation of phospholamban at both serine 16 and threonine 17 leading to a significant inhibition of SR Ca(2+)-ATPase 2A (SERCA2A) and subsequent reduced Ca(2+) uptake into the intracellular Ca(2+) store. Taken together, our data show that Ech A negatively regulates cardiac contractility by inhibiting SERCA2A activity, which leads to a reduction in internal Ca(2+) stores.

Leptin is a 16 kDa pro-satiety peptide produced primarily not only by white adipocytes but also by numerous other tissues including the heart. Circulating leptin exerts its effect through specific receptors, although its principle actions are dependent on the activation of the long form of the leptin receptor, termed OBRb. As leptin is also produced within the cardiomyocyte, we hypothesized that the peptide can also exert effects by targeting intracellular sites. Accordingly, we determined whether cardiac mitochondria express functional leptin receptors. The presence of mitochondrial OBRb was identified through Western blotting of isolated mitochondria, immunofluorescence as well as immunogold labeling with electron microscopy. Although leptin had no direct effect on mitochondrial integrity, it profoundly enhanced the ability of calcium to induce mitochondrial swelling, an effect partially reversed by an OBR antagonist. 24 h exposure to leptin (50 ng/ml) was without effect on mitochondria in cultured neonatal rat ventricular myocytes in contrast to leptin tagged with a 10 amino acid membrane translocation sequence which significantly induced mitochondrial permeability transition pore opening, whereas both leptins produced a hypertrophic response. Our results therefore show that mitochondria express functional OBR which may be of importance toward understanding the role of intracellularly derived leptin in cardiac physiology and pathology.

The influence of intracellular administration of angiotensin (1-7) (Ang 1-7) on the inward calcium current was investigated in myocytes isolated from the left ventricle of Wistar Kyoto rat hearts using the patch-clamp technique. The results indicated: (1) the intracellular administration of Ang (1-7) (100 nM) enhanced the peak inward calcium current (I Ca); (2) the intracellular administration of A779 (100 nM) which a Mas receptor inhibitor, abolished the effect of Ang (1-7) on the calcium current; (3) the activation of PKA and consequent phosphorylation of calcium channels seems to be the mechanism involved in the increment of calcium current induced by the heptapeptide because the intracellular dialysis of the PKA inhibitor suppressed the effect of the heptapeptide; (4) the effect of Ang (1-7) was not related to its secretion into the extracellular space; (5)intracellular dialysis of Ang II (100 nM) has an opposite effect and reduced the peak I Ca; (6) extracellular administration of Ang II (100 nM) to cells previously dialyzed with Ang (1-7) also reduced the peak I Ca previously enhanced by Ang (1-7); and (7) intracellular Ang (1-7) reduced the heart cell volume. Implications for heart contractility were discussed.

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca(2+)]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (I Ca, I Ks, and I Kr), while it is increased by I Na and I to. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action.

SKF-96365 (1-(beta-[3-(4-methoxy-phenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride) is a general TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in cardiovascular system. Recent reports showed that SKF-96365 induced a reduction in cardiac conduction. The present study investigates whether the reduced cardiac conduction caused by SKF-96365 is related to the blockade of voltage-gated sodium current (I Na) in rat ventricular myocytes using the whole-cell patch voltage-clamp technique. It was found that SKF-96365 inhibited I Na in rat ventricular myocytes in a concentration-dependent manner. The compound (1 μM) negatively shifted the potential of I Na availability by 9.5 mV, increased the closed-state inactivation of I Na, and slowed the recovery of I Na from inactivation. The inhibition of cardiac I Na by SKF-96365 was use-dependent and frequency-dependent, and the IC₅₀ was decreased from 1.36 μM at 0.5 Hz to 1.03, 0.81, 0.61, 0.56 μM at 1, 2, 5, 10 Hz, respectively. However, the selective TRPC3 antagonist Pyr3 decreased cardiac I Na by 8.5% at 10 μM with a weak use and frequency dependence. These results demonstrate that the TRPC channel antagonist SKF-96365 strongly blocks cardiac I Na in use-dependent and frequency-dependent manners. Caution should be taken for interpreting the alteration of cardiac electrical activity when SKF-96365 is used in native cells as a TRPC antagonist.

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.

The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.

Correlation of the thickness of the left ventricular posterior wall (LVPWd) with various parameters, including age, gender, weight and height, was investigated in this study using regression models. Multicenter derived database comprised over 4,000 healthy individuals. The developed models were further utilized in the in vitro-in vivo (IVIV) translation of the drug cardiac safety data with use of the mathematical model of human cardiomyocytes operating at the virtual healthy population level. LVPWd was assumed to be equivalent to the length of one-dimensional string of virtual cardiomyocyte cells which was presented, as other physiological factors, to be a parameter influencing the simulated pseudo-ECG (pseudoelectrocardiogram), QTcF and ∆QTcF, both native and modified by exemplar drug (disopyramide) after I Kr current disruption. Simulation results support positive correlation between the LVPWd and QTcF/∆QTc. Developed models allow more detailed description of the virtual population and thus inter-individual variability influence on the drug cardiac safety.

Circulating leptin levels are elevated in obesity and hyperleptinaemia has been postulated to be an independent risk factor for the development of cardiovascular diseases. Although many studies have been published on the mechanisms involved in the effects of leptin on cardiac function and pathological remodeling, scarce information is currently available analyzing the influence of prolonged leptin treatment on ionic cardiac channels remodeling in adult ventricular myocytes. Enzymatically isolated adult rat ventricular myocytes were treated with leptin or vehicle for 48h. Real-Time RT-PCR were used to analyze mRNA expression of Kir2.1, Cav1.2, Cav 3.1, Kv4.2 and Kv4.3 α-subunits and KChIP2 auxiliary subunit. The fast transient outward potassium channels (Itof) α-subunits Kv4.2, Kv4.3 and KChIP2 were analyzed by Western-blot. The fast transient outward potassium current and the action potentials were recorded in isolated myocytes by the whole-cell patch-clamp technique. Leptin treatment induced an up-regulation of Kv4.2, Kv4.3 and KChIP2 subunits mRNA expression. However, transcriptional levels of Kir2.1, Cav1.2, or Cav3.1 α-subunit channels were unmodified by leptin. Protein expression levels of Kv4.2, Kv4.3 and KChIP2 subunits were also increased by leptin. The electrophysiological study showed that leptin increases the fast transient outward potassium current amplitudes and densities shortening action potential duration. In addition, leptin activated Akt signaling in cardiomyocytes and this mechanism was involved in the effect of leptin on Itof channels. In conclusión, leptin increases both the expression and function of Itof channels in adult ventricular myocytes and this mechanism involves Akt signaling. Altogether these data suggest that leptin could exert beneficial or detrimental effects depending on the initial ventricular myocyte repolarizing reserve.

The inward rectifier K⁺ current (IK1) plays an important role in terminal repolarization and stabilization of the resting potential in cardiac cells. Although IK1 was shown to be sensitive to changes in intracellular Ca²⁺ concentration ([Ca²⁺]i), the nature of this Ca²⁺ sensitivity-in spite of its deep influence on action potential morphology-is controversial. Therefore, we aimed to investigate the effects of a nonadrenergic rise in [Ca²⁺]i on the amplitude of IK1 in canine and human ventricular myocardium and its consequences on cardiac repolarization. IK1, defined as the current inhibited by 10 μM Ba²⁺, was significantly increased in isolated canine myocytes following a steady rise in [Ca²⁺]i. Enhanced IK1 was also observed when [Ca²⁺]i was not buffered by ethylene glycol tetraacetic acid, and [Ca²⁺]I transients were generated. This [Ca²⁺]i-dependent augmentation of IK1 was largely attenuated after inhibition of CaMKII by 1 μM KN-93. Elevation of [Ca²⁺]o in multicellular canine and human ventricular preparations resulted in shortening of action potentials and acceleration of terminal repolarization. High [Ca²⁺]o enhanced the action potential lengthening effect of the Ba(2+)-induced IK1 blockade and attenuated the prolongation of action potentials following a 0.3-μM dofetilide-induced IKr blockade. Blockade of IKs by 0.5 μM HMR-1556 had no significant effect on APD90 in either 2 mM or 4 mM [Ca²⁺]o. It is concluded that high [Ca²⁺]i leads to augmentation of the Ba²⁺-sensitive current in dogs and humans, regardless of the mechanism of the increase. This effect seems to be at least partially mediated by a CaMKII-dependent pathway and may provide an effective endogenous defense against cardiac arrhythmias induced by Ca²⁺ overload.

Cardiac CaV1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the CaV1.2 channel is the target of Ca(2+)-dependent and Ca(2+)-independent regulation of the channel. Recent studies on C-terminal truncated forms of the CaV1.2 channel reported neonatal death, reduced CaV1.2 current, and failure of β-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the CaV1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca(2+) currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca(2+) channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the CaV1.2 channel but a twofold increase in the level of mRNA for the CaV1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of CaV1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated CaV1.2 channel with an upregulation of the CaV1.3 channel.

Slow delayed rectifier potassium current (IKs) is important in action potential (AP) repolarization and repolarization reserve. We tested the hypothesis that there are sex-specific differences in IKs, AP, and their regulation by β-adrenergic receptors (β-AR's) using whole-cell patch-clamp. AP duration (APD90) was significantly longer in control female (F) than in control male (M) myocytes. Isoproterenol (ISO, 500 nM) shortened APD90 comparably in M and F, and was largely reversed by β1-AR blocker CGP 20712A (CGP, 300 nM). Inhibition of IKs with chromanol 293B (10 μM) resulted in less APD prolongation in F at baseline (3.0 vs 8.9 %, p < 0.05 vs M) and even in the presence of ISO (5.4 vs 20.9 %, p < 0.05). This suggests that much of the ISO-induced APD abbreviation in F is independent of IKs. In F, baseline IKs was 42 % less and was more weakly activated by ISO (19 vs 68 % in M, p < 0.01). ISO enhancement of IKs was comparably attenuated by CGP in M and F. After ovariectomy, IKs in F had greater enhancement by ISO (72 %), now comparable to control M. After orchiectomy, IKs in M was only slightly enhanced by ISO (23 %), comparable to control F. Pretreatment with thapsigargin (to block SR Ca release) had bigger impact on ISO-induced APD shortening in F than that in M (p < 0.01). In conclusion, we found that there are sex differences in IKs, AP, and their regulation by β-AR's that are modulated by sex hormones, suggesting the potential for sex-specific antiarrhythmic therapy.

Zinc exists in biological systems as bound and histochemically reactive free Zn(2+) in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn(2+) on the L-type Ca(2+) current (I (CaL)) and its modulation by β-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca(2+) ions, Zn(2+) could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca(2+). In the presence of Ca(2+), extracellular Zn(2+) demonstrated strong bimodal inhibitory effects on the I (CaL), with half-inhibition occurring around 30 nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn(2+) also significantly inhibited the I (CaL) with a half-inhibitory effect at 12.7 nM. Moreover, β-adrenergic stimulation was markedly reduced by intracellular Zn(2+) at even lower concentrations (<1 nM) as a consequence of Zn(2+)-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn(2+) modulate transmembrane Ca(2+) movements and their regulation by β-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn(2+) concentration is reduced and the intracellular one is increased, our results help to explain both Ca(2+) overload and marked reduction in the β-adrenergic stimulation in these diseases.

We studied the content of resident myocardial stem cells, cardiomyocyte precursors, in myocardial biopsy specimens from the right ventricular outflow tract of patients of the first two years of life with tetralogy of Fallot. Myocardial resident stem cells were detected by the method of confocal immunohistochemistry using antibodies to c-kit and sarcomeric α-actin. The diameter of right ventricular cardiomyocytes was measured; the presence of myolysis zones was semiquantitatively evaluated. Electron microscopic analysis of right ventricular cardiomyocytes was performed. The data on the content of resident myocardial stem cells were compared with clinical and functional parameters of the patients and morphological peculiarities of the myocardium. C-kit-positive resident myocardial stem cells were detected in the right ventricle of 17.4% patients with tetralogy of Fallot. The content of resident myocardial stem cells in these patients varied from 4 to 45 (median 11) per 1 mln cardiomyocytes; this parameter was higher in patients with high content of small cardiomyocytes (diameter <10 μ) and cardiomyocytes with incomplete myofibril assembly in the right ventricular myocardium.

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na(+) channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca(2+) current (I (Ca)) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I (Ca) in a reversible manner without altering inactivation kinetics of I (Ca). Fitting results to the Hill equation, an IC(50) value of 55 ± 2 μM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 μM nisoldipine, indicating that it was really I (Ca). Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I (Ca), which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I (Ca) and I (Na). This is the first report demonstrating TTX to interact with L-type calcium current in the heart.

Contractile cardiomyocytes in various parts of the heart differ in shape, size, ploidy, and other parameters. However, it is not known whether their population is heterogeneous within each heart chamber. In this paper, dry weight and ploidy of cardiomyocytes were estimated in different parts of rat left ventricle. It was found that the dry weight of cardiomyocytes in medial part of left ventricular anterior wall is higher than in other parts of the ventricle. Cardiomyocyte ploidy is the same in different regions of the left ventricle.

Expression and function of cardiac ion channels exhibit postnatal developmental changes, which, however, has not yet been proven in ventricular myocytes isolated using similar techniques. In this study, ventricular myocytes were enzymatically dissociated from mouse heart at different postnatal ages (including postnatal day 0) by similar techniques using Langendorff perfusion. Whole-cell patch-clamp experiments were performed to record action potentials, I (K1), I (Kr), I (Kur), I (ss), and I (Ca,L), in ventricular myocytes freshly isolated from postnatal days 0, 7, and 14 and adult mice. Viable ventricular myocytes of day-0 mouse heart exhibited spindle-shaped appearance having cell length of approximately 50 μm, which gradually developed to a rod-shaped one having clear cross striation with cell length of approximately 120 μm (adult). The action potential duration markedly shortened, while the resting membrane potential depolarized to a small but significant extent during postnatal development. I (K1) density was maximal in postnatal day-0 ventricular myocytes and gradually decreased during development, which was accompanied by postnatal depolarization of resting membrane potential. However, I (K1) density was markedly decreased by approximately 80% in postnatal day-0 ventricular myocytes, when isolated by the chunk method. Quantitative real-time polymerase chain reaction (PCR) and western blot analyses demonstrated higher Kir2.3 expression but lower expression levels of Kir2.1 and Kir2.2 in day-0 mouse ventricles, compared with those of day-14 and adult mouse ventricles. Whereas I (Kr) exhibited marked decrease during postnatal development, I (Kur), I (ss), and I (Ca,L) exhibited postnatal developmental increase. The present cell isolation method using the Langendorff perfusion thus found that, in mouse ventricles, I (K1) exhibited postnatal developmental decrease, associated with depolarization of resting potential.

Obesity is associated with increased leptin production which may contribute to cardiac hypertrophy. However, the mechanism of leptin-induced cardiac hypertrophy remains incompletely understood. The Rho family (RhoA, Rac1, and Cdc42) and mammalian target of rapamycin (mTOR) have recently emerged as important regulators of cell growth. We therefore explored the roles and interrelationships of phosphatidylinositol 3-kinase (PI3K), mTOR, and the Rho family in the regulation of actin polymerization and leptin-induced hypertrophy in cultured neonatal rat ventricular myocytes. Five minutes treatment with leptin (3.1 nM) resulted in activation of RhoA and Rac1 (by 330 and 160%, respectively, P < 0.05) which was significantly attenuated by AG-490 (50 μM) and LY294002 (10 μM), specific inhibitors of JAK2 and PI3K, respectively. However, Cdc42 activity was unaffected by leptin. The hypertrophic effect of leptin was associated with an increase in phosphorylation of p70(S6K), the major target of mTOR, by 110% (P < 0.05). The specific mTOR inhibitor rapamycin (10 nM) attenuated leptin-induced RhoA and Rac1 activation. Furthermore, the leptin-induced decrease in the G/F-actin ratio, a measure of actin polymerization, was blunted by rapamycin. Leptin produced activation of the transcriptional factor GATA4 which was attenuated by the RhoA inhibitor C3, the p38 MAPK inhibitor SB203580 (10 μM) as well as rapamycin. Our results demonstrate a critical role for PI3K/mTOR/p70(S6K) in leptin-induced RhoA activation resulting in cardiomyocyte hypertrophy associated with GATA4 stimulation.

Electroporation induced by high-strength electrical fields has long been used to investigate membrane properties and facilitate transmembrane delivery of molecules and genes for research and clinical purposes. In the heart, electric field-induced passage of ions through electropores is a factor in defibrillation and postshock dysfunction. Voltage-clamp pulses can also induce electroporation, as exemplified by findings in earlier studies on rabbit ventricular myocytes: Long hyperpolarizations to ≤-110 mV induced influx of marker ethidium and irregular inward currents that were as large with external NMDG(+) as Na(+). In the present study, guinea pig ventricular myocytes were bathed with NMDG(+), Na(+) or NMDG(+) + La(3+) solution (36°C) and treated with five channel blockers. Hyperpolarization of myocytes in NMDG(+) solution elicited an irregular inward current (I (ep)) that reversed at -21.5 ± 1.5 mV. In myocytes hyperpolarized with 200-ms steps every 30 s, I (ep) occurred in "episodes" that lasted for one to four steps. Boltzmann fits to data on the incidence of I (ep) per experiment indicate 50% incidence at -129.7 ± 1.4 mV (Na(+)) and -146.3 ± 1.6 mV (NMDG(+)) (slopes ≈-7.5 mV). I (ep) amplitude increased with negative voltage and was larger with Na(+) than NMDG(+) (e.g., -2.83 ± 0.34 vs. -1.40 ± 0.22 nA at -190 mV). La(3+) (0.2 mM) shortened episodes, shifted 50% incidence by -35 mV and decreased amplitude, suggesting that it inhibits opening/promotes closing of electropores. We compare our findings with earlier ones, especially in regard to electropore selectivity. In the Appendix, relative permeabilities and modified excluded-area theory are used to derive estimates of electropore diameters consistent with reversal potential -21.5 mV.

The influence of cell swelling on cell communication was investigated in cardiomyocytes isolated from the ventricle of adult rats. Measurements of dye coupling were performed in cell pairs using intracellular dialysis of Lucifer Yellow CH. The pipette was attached to one cell of the pair and after a gig ohm seal was achieved, the membrane was ruptured by a brief suction allowing the dye to diffuse from the pipette into the cell. Fluorescence of the dye in the injected as well as in non-dialyzed cell of the pair was continuously monitored. The results indicate that in cell pairs exposed to hypotonic solution the cell volume was increased by about 60% within 35 min and the dye coupling was significantly reduced by cell swelling. Calculation of gap junction permeability (P(j)) assuming an the intracellular volume accessible to intracellular diffusion of the dye as 12% of total cell volume, showed an average P(j) value of 0.16 ± 0.04 × 10(-4) cm/s (n = 35) in the control and 0.89 ± 1.1 × 10(-5) cm (n = 40) for cells exposed to hypotonic solution (P < 0.05). Similar results were found assuming intracellular volumes accessible to the dye of 20 and 30% of total cell volume, respectively. Cell swelling did not change the rate of intracellular diffusion of the dye. The results which indicate that cell volume is an important regulator of gap junction permeability, have important implications to myocardial ischemia and heart failure as well as to heart pharmacology because changes in cell volume caused by drugs and transmitters can impair cell communication with consequent generation of slow conduction and cardiac arrhythmias.

In vivo imaging of the cardiac 3D fibre architecture is still a challenge, but it would have many clinical applications, for instance to better understand pathologies and to follow up remodelling after therapy. Recently, cardiac MRI enabled the acquisition of Diffusion Tensor images (DTI) of 2D slices. We propose a method for the complete 3D reconstruction of cardiac fibre architecture in the left ventricular myocardium from sparse in vivo DTI slices. This is achieved in two steps. First we map non-linearly the left ventricular geometry to a truncated ellipsoid. Second, we express coordinates and tensor components in Prolate Spheroidal System, where an anisotropic Gaussian kernel regression interpolation is performed. The framework is initially applied to a statistical cardiac DTI atlas in order to estimate the optimal anisotropic bandwidths. Then, it is applied to in vivo beating heart DTI data sparsely acquired on a healthy subject. Resulting in vivo tensor field shows good correlation with literature, especially the elevation (helix) angle transmural variation. To our knowledge, this is the first reconstruction of in vivo human 3D cardiac fibre structure. Such approach opens up possibilities in terms of analysis of the fibre architecture in patients.

To address whether adult rat ventricular cardiomyocytes (ARVCs) exposed to oxidant stress die via apoptosis (secondarily by necrosis) or primarily by necrosis, we exposed ARVCs to hydrogen peroxide (H2O2; 0.1-100 microM) for up to 24 h and then compared them with isoproterenol-induced apoptotic and Triton X-induced necrotic controls. Cellular shrinkage preceded plasma membrane disruption, reflected by trypan blue uptake in ARVCs exposed to lower concentrations of H2O2 (<1 microM; an apoptotic pattern), but the order was reversed in cells exposed to higher concentrations of H2O2 (>1 microM; a necrotic pattern). DNA fragmentation, caspase-3 activation, mitochondrial membrane potential preservation, and ATP preservation were all apparent in ARVCs treated with low H2O2 (0.5 microM), but not in those treated with high H2O2 (10 microM). In addition, electron microscopy revealed unique morphology in H2O2-treated ARVCs; i.e., the nuclei had a homogeneous ground glass-like appearance that was never accompanied by chromatin condensation. Apparently, high concentrations of H2O2 caused primary necrosis in ARVCs, whereas low concentrations induced biochemically comparable apoptosis, although the latter did not satisfy the morphological criteria of apoptosis. These findings caution against the use of oxidant stress, H2O2 in particular, as an inducer of apoptosis in ARVCs.

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2DeltaCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin's effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin.

This study examined the influence of two intensities of exercise on ventricular myocyte shortening and intracellular calcium in the streptozotocin (STZ)-induced diabetic rat. Animals were divided into four groups: control sedentary (CS), diabetic sedentary (DS), diabetic light exercise (DLE; 5 x 30 min/week, 9 m/min) and diabetic moderate exercise (DME; 5 x 30 min/week, 18 m/min) and the exercise programme started 2 months after STZ treatment. Time to peak (TPK) shortening was prolonged in myocytes from DS (112.1 +/- 2.5 ms) compared to CS (98.1 +/- 2.1 ms) rats and was not additionally altered by either light (117.0 +/- 2.1 ms) or moderate (115.4 +/- 2.0 ms) exercise. TPK of the Ca(2+) transient was not significantly altered by STZ treatment (69.4 +/- 2.4 ms) but was prolonged by light (79.8 +/- 3.5 ms) and moderate (76.6 +/- 2.9 ms) exercise compared to CS (65.5 +/- 2.7 ms). Data from this study suggest that the chosen intensities of exercise were ineffective in modulating the dynamics of cardiac muscle contraction and reversing the deleterious effects of diabetes on heart-muscle contraction.

We have previously described a model to engineer three-dimensional (3-D) heart muscle in vitro. In the current study, we extend our model of 3-D heart muscle to engineer a functional cell-based cardiac pressure generating construct (CPGC). Tubular constructs were fabricated utilizing a phase separation method with chitosan as the scaffolding material. Primary cardiac cells isolated from rat hearts were plated on the surface of fibrin gels cast in 35 mm tissue culture dishes. CPGCs (N = 8) were formed by anchoring the tubular constructs to the center of the plate with primary cardiac cells seeded in fibrin gels wrapped around the tubular constructs. Intraluminal pressure measurements were evaluated with and without external electrical stimulation and histological evaluation performed. The fibrin gel spontaneously compacted due to the traction force of the cardiac cells. By 14 d after original cell plating, the cardiac cells had completely formed a monolayer around the tubular construct resulting in the formation of a cell-based CPGC. The spontaneous contractility of the CPGC was macroscopically visible and resulted in intraluminal pressure spikes of 0.08 mmHg. Upon electrical stimulation, the CPGCs generated twitch pressures of up to 0.05 mmHg. In addition, the CPGC constructs were electrically paced at frequencies of up to 3 Hz. Histological evaluation showed the presence of a continuous cell monolayer around the surface of the tubular construct. In this study, we describe a novel in vitro method to engineer functional cell-based CPGCs and demonstrate several physiological metrics of functional performance.

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).

The content of colony-forming cells in myocardial cell culture from the perinecrotic zone of rat heart was evaluated on day 40 after cryodestruction. The mean cellularity after cryodestruction was 12-fold lower than in intact animals. Intramyocardial transplantation of bone marrow cells (mononuclears, mesenchymal stem cells, and mesenchymal stem cells treated with 5-azacitidine) into the perinecrotic zone increased the content of colony-forming cells. After transplantation of mesenchymal stem cells and mesenchymal stem cells treated with 5-azacitidine, the number of colonies reached 33 +/- 7 and 11 +/- 3, the mean cellularity being 2975 +/- 80 and 1105 +/- 42 cells/cm2, respectively. Hence, intramyocardial transplantation of mesenchymal stem cells created an appreciable pool of colony-forming cells in the myocardial perinecrotic zone. Treatment with 5-azacitidine reduced survival of mesenchymal stem cells after intramyocardial transplantation.

Intercellular adhesion molecule-1 plays a key role in mediating inflammatory and immune responses. There is also increasing appreciation of the role of its soluble form, sICAM-1, in regulating inflammation. This study evaluated the effects of hypoxia and N-acetyl-L-cysteine on sICAM-1 production by adult rat cardiac fibroblasts. By ELISA, hypoxia was found to cause a 61% increase in sICAM-1 in cardiac fibroblast culture supernates. However, RT-PCR did not reveal a concomitant increase in cell surface ICAM-1 transcript levels, suggesting that the increase in sICAM-1 may involve post-transcriptional and/or post-translational mechanisms. Using pharmacological inhibitors, it was observed that p42/44 MAPK and PKC mediate the stimulatory effect of hypoxia on sICAM-1 production. Remarkably, N-acetyl-L-cysteine caused a 3-fold increase in sICAM-1 by p42/44 MAPK-, p38 MAPK- and PKC-independent mechanisms. Pyrrolidine dithiocarbamate, another potent antioxidant, also augmented sICAM-1. The findings presented in this communication underscore the link between redox status and sICAM-1 release from cardiac fibroblasts. Further, because hypoxia is a major component of myocardial ischemia and is pro-inflammatory, and both N-acetylcysteine and pyrrolidine dithiocarbamate are clinically used antioxidants, the observations may have clinical significance.