Ventricular Myocytes Of Neonatal Rats Research Articles

Calcineurin Aβ gene knockdown inhibits transient outward potassium current ion channel remodeling in hypertrophic ventricular myocyte.

It has been shown that the activation of calcineurin is involved in regulating ion channel remodeling in hypertrophic cardiomyocytes. But the precise role of calcineurin in the regulation of transient outward potassium current (I to), an ion channel associated with fatal arrhythmia, remains controversial. This study aimed to examine the effects of calcineurin Aβ (CnAβ) gene knockdown on I to channel remodeling and action potential duration (APD) in the hypertrophic ventricular myocytes of neonatal rats. Results showed that phenylephrine stimulation caused hypertrophy of ventricular myocytes, upregulation of CnAβ protein expression, downregulation of Kv4.2 mRNA and protein expression, a decrease in I to current density, and prolongation of APD. CnAβ gene knockdown significantly inhibited the effects of phenylephrine stimulation. Our data indicate that CnAβ gene knockdown can inhibit I to channel remodeling and APD prolongation in hypertrophic neonatal rat ventricular myocytes. This finding suggests that calcineurin may be a potential target for the prevention of malignant ventricular arrhythmia in a hypertrophic heart.

Suppression of Oxidative Stress and Apoptosis in Electrically Stimulated Neonatal Rat Cardiomyocytes by Resveratrol and Underlying Mechanisms.

We explored the effects of resveratrol on oxidative stress in cardiomyocytes subjected to rapid electrical stimulation (RES) and also investigated the underlying mechanisms. Cultured ventricular myocytes of neonatal rat were subjected to RES at 4.0 Hz, with or without resveratrol, an NADPH oxidase inhibitor apocyanin (APO) or a Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor autocamtide-2-inhibitory peptide (AIP). Cell counts, to optimize resveratrol concentration, and angiotensin II content were evaluated. Reactive oxygen species (ROS), intracellular Ca in cardiomyocytes, and cardiomyocyte apoptosis were also assessed. Levels of methionine sulfoxide reductase A (MsrA), Nox, oxidative CaMKII (OX-CaMKII), and cleaved caspase-3 in cardiomyocytes were examined. Resveratrol treatment, as compared with APO and AIP, significantly decreased ROS levels, improved Ca amplitudes, and intracellular Ca transient decay rates, and inhibited cardiomyocyte apoptosis. Resveratrol also increased MsrA protein levels. In cardiomyocytes subjected to RES, after pretreatment with resveratrol or APO, protein levels of Nox4, Nox2, OX-CaMKII, and cleaved caspase-3 were decreased. In comparison, with AIP pretreatment, only Nox2, OX-CaMKII, and cleaved caspase-3 were decreased. However, in the presence of dimethyl sulfoxide, a competitive inhibitor of MsrA function, a decrease in cleaved caspase-3 did not occur. Resveratrol decreased ROS, partially through the inhibition of NADPH oxidase activity and upregulation of MsrA expression.

Amelioration of Chronic Intermittent Hypobaric Hypoxia on DecreasedL-type Ca^(2+) Currents by Ischemia/Reperfusion in Cardiomyocytes ofDeveloping Rat

The aim of the present study was to observe the effect of chronic intermittent hypobaric hypoxia (CIHH) on L-type calcium channel current (I_(CaL)) in developing rat in order to investigate the ionic mechanism of the car-dioprotective effect of CIHH. Age- and body weight-matched postnatal male Sprague-Dawleyrats were divided into four groups: 28-day CIHH group (CIHH28), 42-day CIHH group (CIHH42), 28-day control group (CON28) and 42-day control group (CON42). CIHH neonatal rats were experienced a CIHH treatment simulating 3000-m altitude (P_B = 525 mmHg, P_(O2) = 108.8 mmHg, 5 h/day) in a hypobaric chamber for 28 and 42 days, respectively. Whole-cell patch clamp technique was used to record I_(CaL) in ventricular myocytes of neonatal rats. The results showed: There were no differences of peak current, current-voltage (I-V) relationship curve, and dynamics of I_(CaL) between CON and CIHH cardiomyocytes under normal external solution (P ＞ 0.05). The peak I_(CaL) was decreased and I-V relationship curve was shifted upward during simulated ischemia/reperfusion (I/R) (P ＜ 0.05), but the change of I_(CaL) was much smaller in CIHH cardiomyocytes than in CON cardiomyocytes (P ＜ 0.05). The steady-state inactivation curve of I_(CaL) was shifted to the left during I/R in CON28 and CON42 cardiomyocytes (P ＜ 0.05), but not in CIHH28 and CIHH42 cardiomyocytes (P ＞ 0.05). In conclusion, CIHH has no effect on I_(CaL) in ventricular myocytes of developing rat under basic condition, but effectively antagonizes the inhibition of simulated I/R on I_(CaL), which might be one of the ionic mechanisms for CIHH cardiac protection against I/R injury in developing rats.

Upregulation of neuropeptide Y in the cardiac sympathetic nerves causes stress (takotsubo) cardiomyopathy

Background: Abrupt conditional changes can interfere with the harmony between the brain and the heart, thereby following impaired cardiovascular function. Stress cardiomyopathy (SC) is a typical sample. SC is a disorder associated with transient left ventricular apical ballooning that is induced by stress. The precise molecular mechanisms of SC remain unclear. Methods and results: (1) By analyzing a new animal model of SC in rodents inducing by epilepsy, we demonstrated hypothalamic activation due to Ccl2 causes LV dysfunction like SC. (2) Upstream sympathetic activation induces strong upregulation of neuropeptide Y (NPY) expression in the left stellate ganglion (LSG) and LV sympathetic nerves. (3) NPY reduced the frequency of Ca2+ sparks when co-injected with noradrenaline in the ventricular myocytes of adult rat. (4) We demonstreted that NPY reduced the contraction of ventricular myocytes of neonatal rat when co-incuvated with noradrenaline by using phase-contrast microscopy. (5) NPY injection into the LSG induces SC-like LV wall motion. (6) The incidence of SC was less frequent in NPY-/- mice than in NPY+/+ mice. Conclusions: Our results demonstrate how brain activation translates into molecular signals in the cardiac nervous system and leads to LV apical ballooning.

Roles of 3,4-methylenedioxymethamphetamine (MDMA)-induced alteration of connexin43 and intracellular Ca2+ oscillation in its cardiotoxicity

Although it is well known that 3,4-methylenedioxymethamphetamine (MDMA) can cause various cardiovascular abnormalities and even sudden death from cardiac arrhythmia, whether it has any effect on myocardial gap junctions, which might be one of the targets mediating MDMA-induced cardiotoxicity, remains unclear. ObjectiveTo test the hypothesis that MDMA may affect the myocardial gap junction protein connexin43 (Cx43) and induce cardiac dysrhythmia. Method(1) In vivo study: adult rats were treated with a single dose MDMA administration (20mg/kg, i.p.). Electrocardiogram detection and immunohistochemical analysis were performed to evaluate cardiac function and expression of Cx43, respectively; (2) in vitro study: cultured ventricular myocytes of neonatal rats were treated with MDMA (10, 100, 1000μmol/L) for 1h. Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to investigate the total Cx43 mRNA expression. Immunofluorescent analysis was used to evaluate the amount of junctional Cx43. The phosphorylation status of Cx43 at site Ser368 and intracellular Ca2+ oscillation were also studied. ResultsObvious changes in electrocardiographic patterns were found in rats following MDMA administration. They were characterized by prolonged QRS duration associated with increased amplitude of QRS complex. The heart rates in treated rats were significantly decreased compared to the rats in the control group. The immunohistochemical findings revealed a significant decrease in Cx43 expression. The in vitro study also showed a marked decline in total Cx43 protein associated with reduction of Cx43 mRNA, whereas the phosphorylated Cx43 at Ser368 was increased. Decrease of junctional Cx43 was found correlated with reduction in N-cadherin induced by high concentration of MDMA. Additionally, confocal microscopy findings revealed alteration of intracellular calcium oscillation patterns characterized by high frequency and increasing influx Ca2+. ConclusionsMDMA reduces expression of cardiac gap junction protein Cx43. The increase of phosphorylation status of Cx43 at Ser368 induced by MDMA is attributed, at least in part, to the Ca2+-dependent regulation of protein kinase C (PKC) activity. Our findings provide first evidence of MDMA-mediated changes in those cardiac gap junctions that may underlie MDMA-induced cardiac arrhythmia.

The effect of Intermedin on Angiotensin II and Endothelin-1 Induced Ventricular Myocyte Hypertrophy in Neonatal Rat

Intermedin (IMD), a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), may have localized actions as a modulator of cardiac function. The aim of the study is to explore the effect of IMD on angiotensin II (Ang II) and endothelin-1 (ET-1) induced hypertrophy in ventricular myocytes of neonatal rat and to try to elucidate the possible mechanism. Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or ET-1 (60 micromol/L) in the presence and absence of IMD (1 micromol/L). Hypertrophic responses (including cell surface area, alpha-actin, and beta-myosin heavy chain mRNA expression) and cardiomyocyte expression of NADPH oxidase gp91phox were determined. Ang II induced increases in cardiomyocyte size to 305 +/- 32 microm2 (n = 198, p < 0.05, at 48 hours), alpha-actin expression to 4 +/- 2.8-fold (n = 6, p < 0.05, at 48 hours) and beta-myosin heavy chain expression to 11 +/- 4.8-fold (n = 6, p < 0.05, at 48 hours), and expression of the gp91phox subunit of NADPH oxidase to 29.4 +/- 12.7-fold (n = 6, p < 0.05, at 48 hours). These effects were all significantly inhibited by IMD; cardiomyocyte size, alpha-actin expression, beta-myosin heavy chain expression, and gp91phox expression were reduced to 265 +/- 32 microm2 (n = 374, p < 0.05), 3.0 +/- 1.7-fold (n = 6, p < 0.05), 8.7 +/- 4.9-fold (n = 6, p < 0.05), 3.9 +/- 3-fold (n = 6, p < 0.05), respectively. IMD also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation. IMD exerts an antihypertrophic effect on neonatal cardiomyocytes by reduced levels of superoxide, suggesting that an antioxidant action contributes to the antihypertrophic actions of IMD.

English

Aconitine, a strong poisonous type of alkaloid, has a pharmaceutical effect in stimulating the membranes of cardiomyocyte. However, other effects of aconitine on the Connexin43 (Cx43) and PKC&alpha; expression&nbsp;on cardiomyocyte are unknown. In this study, we investigated whether aconitine also mediates the phosphorylation status of Cx43 and PKC&alpha;&nbsp;in cultured ventricular myocytes of neonatal rats. The band intensity of phosphorylated Cx43 and nonphosphorylated Cx43 in cultured and aconitine-treated cardiomyocytes were determined by Western blot analysis. The changes in phosphorylation status occurring in PKC&alpha; in cultures were revealed by quantitative immunofluorescent microscopy. A decreased band intensity (0.37&plusmn;0.04) of phosphorylated Cx43 (P-Cx43) and a concomitant increased band intensity (3.56 &plusmn; 0.65)&nbsp;of nonphosphorylated Cx43 (NP-Cx43) were found, compared to the controls (1.00&nbsp;for P-Cx43 and NP-Cx43). It also revealed that, after aconitine treatment, the amount of phosphorylated PKC&alpha; (P-PKC&alpha;) decreased significantly. Similar changes were revealed in phosphorylation status occurring in PKC&alpha; in the cultures under the same treatment conditions. These observations suggest that aconitine not only induces dephosphorylation of Cx43, but also alters expression of P-PKC&alpha; in cultured cardiomyocytes. &nbsp; Key words:&nbsp;Aconitine, cardiomyocyte, connexin 43 (Cx43), protein kinase C-&alpha; (PKC&alpha;), protein phosphorylation.

δ‐OPIOID RECEPTOR STIMULATION ENHANCES THE GROWTH OF NEONATAL RAT VENTRICULAR MYOCYTES VIA THE EXTRACELLULAR SIGNAL‐REGULATED KINASE PATHWAY

1. The aims of the present study were to determine whether delta-opioid receptor stimulation enhanced proliferation of and to investigate the role of the extracellular signal-regulated kinase (ERK) pathway in ventricular myocytes from neonatal rats. 2. At concentratins ranging from 10 nmol/L to 10 micromol/L, [D-Ala2,D-Leu5]enkephalin (DADLE) concentration-dependently promoted myocardial growth and DNA synthesis and altered the cytoskeleton. 3. At 1 micromol/L, DADLE also increased the expression and phosphorylation of ERK. 4. These effects of 1 micromol/L DADLE were abolished by 10 micromol/L naltrindole, a selective delta-opioid receptor antagonist, 10 nmol/L U0126, a selective ERK antagonist, 1 micromol/L staurosporine, an inhibitor of protein kinase (PK) C, and 100 micromol/L Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), an inhibitor of PKA. 5. In conclusion, delta-opioid receptor stimulation enhances the proliferation and development of the ventricular myocytes of neonatal rats. The ERK pathway and related signalling mechanisms, namely PKC and PKA, are involved.

Aconitine alters connexin43 phosphorylation status and [Ca 2+] oscillation patterns in cultured ventricular myocytes of neonatal rats

Aconitine, a highly poisonous type of alkaloid, has a widespread effect in stimulating the membranes of cardiomyocyte. However, other effects of aconitine on cardiomyocyte are unknown. In this study, we investigated whether aconitine also affects the phosphorylation status of connexin43 (Cx43) and intracellular [Ca 2+] oscillation patterns in cultured ventricular myocytes of neonatal rats. As determined by Western blot analysis, a decreased percentage (47.68 ± 2.29%) of phosphorylated Cx43 (P-Cx43) and a concomitant increased percentage (52.32 ± 2.29%) of nonphosphorylated Cx43 (NP-Cx43) were found in aconitine-treated cultures, compared to the controls (82.77 ± 2.04% for P-Cx43 and 17.23 ± 2.04% for NP-Cx43). Quantitative immunofluorescent microscopy revealed similar changes in phosphorylation status occurring in Cx43 containing gap junctions in the cultures under the same treatment conditions. Real-time laser scanning microscopy indicated that intracellular [Ca 2+] oscillations were relatively stable in control cultures, with occasional calcium sparks; after being treated with aconitine, high frequency [Ca 2+] oscillations emerged, whereas typical calcium sparks disappeared. Furthermore, Western blot analysis revealed that, after aconitine treatment, the amount of phosphorylated PKCα decreased significantly. These observations suggest that aconitine not only induces dephosphorylation of Cx43 and PKCα, but also alters intracellular [Ca 2+] oscillation patterns in cultured cardiomyocytes.

ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation.

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

The direct effects of diazepam and midazolam on myocardial depression in cultured rat ventricular myocytes.

We examined the direct myocardial depressant effects of diazepam and midazolam and determined whether a benzodiazepine receptor antagonist, flumazenil, or an L-type Ca2+ channel agonist, Bay K 8644, affects the myocardial depression induced by diazepam and midazolam in cultured rat ventricular myocytes. Ventricular myocytes of neonatal rats were obtained by enzymatic digestion with collagenase and cultured for 6-7 days. The myocytes were stabilized in serum-free medium, and the spontaneous beating rate and amplitude were measured by determining displacement with a fiberoptic sensor. Myocytes were exposed to either diazepam or midazolam at concentrations of 0.1, 1, 10, and 100 microM. The beating rate and amplitude were measured 4 min after diazepam or midazolam administration. In other cells, either diazepam or midazolam was administered at each concentration in the presence of flumazenil or Bay K 8644. Midazolam and diazepam decreased the beating rate and amplitude concentration-dependently. These myocardial depressant effects were prevented by Bay K 8644 and, to a lesser degree, by flumazenil. Thus, the L-type Ca2+ channel is important in the direct myocardial depression caused by diazepam and midazolam. This study describes the direct effect of midazolam and diazepam on intrinsic myocardial contraction using cultured rat ventricular heart cells. Both of these drugs have a direct myocardial depressant effect at the cellular level, which is mainly mediated by an inhibition of the sarcolemmal L-type Ca2+ channel.

The Uncoupling Effect of Diacylglycerol on Gap Junctional Communication of Mammalian Heart Cells Is Independent of Protein Kinase C

Possible regulatory effects on cell-to-cell communication of a synthetic diacylglycerol, an activator of protein kinase C (PKC), were examined in pairs of synchronously beating ventricular myocytes of neonatal rats in primary culture. Junctional communication was estimated by measuring either the rate constant of dye diffusion, with the fluorescence recovery after photobleaching technique, or the cell-to-cell electrical conductance with a double whole-cell voltage clamp. The addition of a freshly prepared emulsion of 1-oleoyl-2-acetyl- sn-glycerol (OAG, 100 μg/ml), either in the bath or in the solution filling the patch pipet, was seen to interrupt intercellular communication within approximately 8 to 10 min. This effect is neither mimicked by stimulation of PKC by a phorbol ester, nor prevented by PKC inhibitors, making it unlikely that, in these cells, PKC activation could induce intercellular uncoupling. During OAG exposures, the intracellular calcium concentration was very modestly increased (by a factor 1.5 to 2), which does not suffice to account for uncoupling. OAG might trigger interruption of cell-to-cell communication by a mechanism analogous to that of other lipophilic molecules (such as aliphatic alcohols or long chain unsaturated fatty acids) which interfere with gap junctions.

Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characterization.

A culture method was developed that permits patterning of the growth of ventricular myocytes of neonatal rats. Regions were created on the culture substrate that either prevented (photoresist coat) or supported (glass surface) attachment of cells. In this way the geometry of interconnecting growth channels could be specified. Single-layered myocyte strands of variable length and with widths of as little as 65 micron (three to four cells wide) were obtained. The shape and orientation of the individual myocytes were a function of growth-channel width: the narrower the channel, the more elongated the cells and the more likely was the long axis to be oriented along the channel axis. In channels with width of 100 micron or less, cells were aligned longitudinally and cross-striated as in vivo. A high degree of morphological cell differentiation required the presence of contractile activity. Maximal diastolic potential (-71 mV), action potential amplitude (93 mV), and maximal upstroke velocity (140 V/sec) did not change with increasing culture age. Mean longitudinal conduction velocity was 0.39 m/sec. No electrophysiological or morphological evidence of photoresist toxicity was seen, and the data indicate a high degree of cell differentiation in the patterned cell cultures. The method thus is suitable for the study of the relation between impulse propagation and structure at a cellular level in artificial networks of predefined shape.

Inhibition of cardiac Na+ currents by isoproterenol

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

Asymmetric charge movement and calcium currents in ventricular myocytes of neonatal rat.

1. Calcium and sodium currents and non-linear capacitive currents were recorded from isolated ventricular cells from neonatal rats, using the whole-cell patch-clamp technique, usually with a holding potential of -100 mV. 2. When recording with internal and external solutions designed to suppress virtually all ionic currents except the calcium current, careful subtraction of all linear capacitive and ionic currents revealed that depolarizations elicited a small transient outward current which preceded the inward calcium current. This outward current was discernible just below the threshold potential for the calcium current and increased with larger depolarizations to a maximum for potentials of about +30 mV and above. 3. Elimination of the calcium current revealed that at each potential the transient outward current was accompanied by a roughly equal transient inward current upon repolarization. The properties of these currents indicate that they are non-linear capacitive currents. Best-fit Boltzmann curves of the 'on' charge (integral of the transient outward current) gave values for qmax, V and k of 3.9 nC/microF, -29.3 mV and 15.5 mV with internal Cs+. The maximum 'on' charge is similar to that found with calcium currents (4.3 nC/microF). Similar values were obtained with internal TEA+. 4. Boltzmann fits of conductance vs. voltage for the calcium channel gave mean values of -15.5 and 13.3 mV for V and k (with internal Cs+); the corresponding values for the sodium channel were -49.9 and 5.4 mV. 5. Pre-pulses (20 ms) to -60 mV inactivated 77% of the peak sodium current, but only inactivated about 10% of the peak calcium current and reduced the maximum 'on' charge (moved at potentials positive to -60 mV) by 19%. 6. With a holding potential of -100 mV, 10 microM-nifedipine blocked 89% of the calcium current, but had little effect on the amount of 'on' charge. The 'off' charge appeared to be slower in the presence of nifedipine. 7. These results and consideration of the number of calcium channels and high-affinity binding sites for dihydropyridines (DHP), suggest that a large part of the charge movement may be related to DHP binding sites and involved with gating calcium channels. Comparison with skeletal muscle suggests similarities in the mechanisms involved in excitation-contraction coupling.

Stretch activated ion channels in ventricular myocytes.

Patch-clamp recordings from ventricular myocytes of neonatal rats identified ionic channels that open in response to membrane stretch caused by negative pressures (1 to 6 cm Hg) in the electrode. The stretch response, consisting of markedly increased channel opening frequency, was maintained, with some variability, during long (greater than 40 seconds) stretch applications. The channels have a conductance averaging 120pS in isotonic KCl, have a mean reversal potential 31 mV depolarized from resting membrane potential, and do not require external Ca++ for activation. The channels appear to be relatively non-selective for cations. Since they are gated by physiological levels of tension, stretch-activated channels may represent a cellular control system wherein beat-to-beat tension and/or osmotic balance modulate a portion of membrane conductance.