Rat Neonatal Myocytes Research Articles

Effects of IGF-1 onIKandIK1Channels via PI3K/Akt Signaling in Neonatal Cardiac Myocytes

Previous studies suggest that sarcolemmal potassium currents play important roles in cardiac hypertrophy. IGF-1 contributes to cardiac hypertrophy via activation of PI3K/Akt signaling. However, the relationships between IGF-1, PI3K/Akt signaling and sarcolemmal potassium currents remain unknown. Therefore, we tested the hypothesis that IGF-1 and PI3K/Akt signaling, independently, decrease sarcolemmal potassium currents in cardiac myocytes of neonatal rats. We compared the delayed outward rectifier (IK) and the inward rectifier (IK) current densities resulting from IGF-1 treatments to those resulting from simulation of PI3K/Akt signaling using adenoviral (Ad) BD110 and wild-type Akt and to those resulting from inhibition of PI3K signaling by LY294002. Ad.BD110 and Ad.Akt decreased IK and these decrements were attenuated by LY 294002. The IGF-1 treatments decreased both IK and IK1 but only the IK decrement was attenuated by LY294002. These findings demonstrate that IGF-1 may contribute to cardiac hypertrophy by PI3K/Akt signal transduction mechanisms in neonatal rat cardiomyocytes. Failure of LY294002 to effectively antagonize IGF-1 induced decrements in IK1 suggests that a signal pathway adjunct to PI3K/Akt contributes to IGF-1 protection against arrhythmogenesis in these myocytes. Our findings imply that sarcolemmal outward and inward rectifier potassium channels are substrates for IGF-1/PI3K/Akt signal transduction molecules.

Cardioplegia and Angiotensin II Receptor Antagonists Modulate Signal Transducers and Activators of Transcription Activation in Neonatal Rat Myocytes

Previous investigations have shown that the signal transducers and activators of transcription (STATs) signaling pathway play an important role in the modulation of apoptosis after ischemia and reperfusion. The mechanism for this enhanced cardioprotection is unknown, but we believe that alterations STATs may play a role. To investigate this hypothesis, we examined the effects of angiotension II type 1 (AT1) and angiotension II type 2 (AT2) receptor antagonist added to cardioplegia on the downstream response of different STATs, connected with proinflammatory pathways (STAT2, STAT5) and prohypertrophic and antiapoptotic pathways (STAT3). Isolated, nonworking hearts (n = 3 per group) from neonatal rats were perfused aerobically (4°C) for 20 min in the Langendorff mode with the modified St. Thomas' Hospital no. 2 (MSTH2) cardioplegic solution (Group 1), the MSTH2 cardioplegic solution + AT1 receptor antagonist (Group 2), and MSTH2 cardioplegic solution + AT2 receptor antagonist (Group 3). Thus, myocytes were isolated by enzymatic digestion, and STAT2, STAT3, and STAT5 were investigated in Western blot studies. Times to arrest after cardioplegia were 8-12 s for all groups. Total cardioplegia delivery volume was about 300 mL for the 20 min. Perfusion with the MSTH2 cardioplegic solution supplemented with AT1 receptor antagonist (Group 2) induced a significant reduction in STAT2 and STAT5 tyrosine phosphorylation (-58 and -63%, respectively, vs. Group 1, P < 0.05). Conversely, STAT2 and STAT5 activation were unaffected by perfusion with the MSTH2 cardioplegic solution supplemented with AT2 receptor antagonist (Group 3). The decreased activation of STAT2 and STAT5 observed in Group 2 was accompanied by reduction of interleukin-1β (-57% in Group 2 vs. Group 1, P < 0.05). There were no significant differences in STAT3 phosphorylation among all groups. Only the addition of AT1 receptor antagonist to MSTH2 cardioplegia significantly decreases the inflammatory response of the neonatal rat cardiomyocytes without affecting antiapoptotic influence provided by tyrosine phosphorylation of STAT3. AT1 receptor antagonist added to cardioplegia represents an additional modality for enhancing myocardial protection during cardiac surgery and could contribute to optimize the ischemia tolerance of the pediatric heart.

Ionic mechanism of shock-induced arrhythmias: role of intracellular calcium.

Strong electrical shocks can cause focal arrhythmias, the mechanism of which is not well known. Strong shocks have been shown to produce diastolic Ca(i)(2+) increase, which may initiate focal arrhythmias via spontaneous Ca(i)(2+) rise (SCR), activation of inward Na(+)/Ca(2+) exchange current (I(NCX)), and rise in membrane potential (V(m)). It can be hypothesized that this mechanism is responsible for generation of shock-induced arrhythmias. The purpose of this study was to examine the roles of SCRs and I(NCX) in shock-induced arrhythmias. The occurrence of SCRs during shock-induced arrhythmias was assessed in neonatal rat myocyte cultures. Simultaneous V(m)-Ca(i)(2+) optical mapping at arrhythmia source demonstrated that V(m) upstrokes always preceded Ca(i)(2+) transients, and V(m)-Ca(i)(2+) delays were not different between arrhythmic and paced beats (5.5 ± 0.9 and 5.7 ± 0.4 ms, respectively, P = .5). Shocks caused gradual rise of diastolic Ca(i)(2+) consistent with membrane electroporation but no significant Ca(i)(2+) rises immediately before V(m) upstrokes. Application of the Ca(i)(2+) chelator BAPTA-AM (10 μmol/L) decreased the duration of shock-induced arrhythmias whereas application of the I(NCX) inhibitor KB-R7943 (2 μmol/L) increased it, indicating that, despite the absence of SCRs, changes in Ca(i)(2+) affected arrhythmias. It is hypothesized that this effect is mediated by Ca(i)(2+) inhibition of outward I(K1) current and destabilization of resting V(m). The possible role of I(K1) was supported by application of the I(K1) inhibitor BaCl(2) (0.2 mmol/L), which increased the arrhythmia duration. Shock-induced arrhythmias in neonatal rat myocyte monolayers are not caused by SCRs and inward I(NCX). However, these arrhythmias depend on Ca(i)(2+) changes, possibly via Ca(i)(2+)-dependent modulation of outward I(K1) current.

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of beta-adrenoceptor activiation, thus, our findings suggest long-term repeated use of beta-adrenergic drugs may induce unexpected functional alteration of VDCCs.

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.

P21-Activated Kinase-1 and Fty720 Alter Atrial Excitation-Contraction Coupling

We previously reported that Pak1 participates in the integrated regulation of excitation-contraction coupling (e-cc) in adult rat ventricular myocytes via activation of protein phosphatase 2A (PP2A). Pak1 inhibited the ability of isoproterenol (ISO) to increase the peak of the Ca2+ transient in a single myocyte without altering the increase in shortening. Here we test the hypothesis that Pak1 signaling regulates atrial e-cc and potentiates arrhythmicity. Under normal conditions single adult rat atrial myocytes expressing constitutively active Pak1 (CA-Pak1) and paced at 0.5 Hz have a slower time constant of [Ca2+ ]i-transient decay compared to uninfected controls as assessed by confocal microscopy and fluo-4 fluorescence. Increase of the pacing frequency to 2.0 Hz resulted in a reduced peak of the [Ca2+ ]i-transient, an increase in diastolic [Ca2+ ]I, and more rapid [Ca2+ ]i decay compared to 0.5 Hz for both CA-Pak1 expressing myocytes and controls. Return to 0.5 Hz pacing restored these values to near controls, with some extra-systolic Ca2+ release. Stimulation with ISO (100 nM) of CA-Pak1-expressing myocytes paced at 3.0 Hz caused 20% missed beats that were subsequently restored on washout. Uninfected myocytes paced at 2.0 Hz in the presence of ISO had 1:1 fidelity of beating to stimulation, however treatment with fingolimod (FTY720, 25 nM) in addition to ISO caused 50% missed beats that were restored on washout of the drug. Cultured monolayers of rat neonatal myocytes expressing CA-Pak1 had a more rapid rate of spontaneous beating than controls that was resistant to further increase with ISO, as assessed by microelectrode array (MEA). We conclude that Pak1 and FTY720 act through the same pathways in atrial myocytes and induce arrhythmic activity under conditions of stress.

Calibrated micropost arrays for biomechanical characterisation of cardiomyocytes

The study of biomechanics of isolated cardiomyocytes can allow us to understand the cardiac function and disease development in the absence of viscoelastic or contractile properties of the surrounding tissue. However, popular techniques such as micropipettes and carbon fibre-based measurements require serial, single-cell measurements and limit the amount of data passing through a system or process. The authors utilise elastomer micropost arrays microfabricated by a replica molding technique for precise and quantitative force measurements of cardiomyocytes with the potential for high throughput. The authors also present a calibration system using a piezoresistive force sensor and video-analysis technique to improve the resolution and validate the analysis of these low-force measurements. Calibrated microposts arrays are applied to measure the contractile forces of rat neonatal cardiomyocytes. Using image processing, the contractile forces for a sample of cardiomyocytes are extracted. It was found that isolated rat neonatal myocytes generate 39±5 nN average contractile force per post and an integrated axial contractile force of 189±20 nN, which is four times smaller than isolated adult Wistar rat cardiomyocytes and 30 times smaller than isolated adult rat cardiomyocytes. With calibration and quantitative image analysis, this study demonstrates that micropost systems can provide precise high-throughput test beds for myocyte mechanics using pharmacologic, small peptide, gene therapies or heart disease models.

Targeted intracellular catalase delivery protects neonatal rat myocytes from hypoxia-reoxygenation and ischemia-reperfusion injury

Hypoxia followed by reoxygenation and ischemia reperfusion cause cell death in neonatal rat ventricular myocytes primarily through the generation of oxidative stress. Extracellular catalase has not been effective in reducing or eliminating ischemia reperfusion- or hypoxia-reoxygenation-induced cell death due both to extracellular degradation and to poor cellular uptake. (1) To determine whether a cell-penetrating catalase derivative with enhanced peroxisome targeting efficiency (catalase-SKL) increases intracellular levels of the antioxidant enzyme in neonatal rat ventricular myocytes; and (2) to determine whether catalase-SKL protects against both hypoxia-reoxygenation and ischemia reperfusion injury. Neonatal rat ventricular myocytes were subjected to 3 or 6 h of hypoxia-reoxygenation or to 1 h of ischemia reperfusion. Extracellular catalase concentration, activity, and subcellular distribution were determined using standard techniques. Reactive oxygen species and related oxidative stress were visualized using 2',7'-dichlorofluorescin diacetate. Cell death was measured using trypan blue exclusion or lactate dehydrogenase release assays. Extracellular catalase activity was higher in (catalase-SKL) transduced myocytes, was concentrated in a membranous cellular fraction, and potently inhibited oxidative stress. In contrast to nontransducible (unmodified) extracellular catalase, catalase-SKL-treated myocytes were protected against both hypoxia-reoxygenation and ischemia reperfusion. (1) Catalase-SKL increased myocyte extracellular catalase content and activity and dramatically increased resistance to hydrogen peroxide-induced oxidation; (2) catalase-SKL protects against both hypoxia-reoxygenation and ischemia reperfusion; (3) catalase-SKL may represent a new therapeutic approach to protect hearts against myocardial hypoxia-reoxygenation or ischemia reperfusion.

Enhanced Fibroblast–Myocyte Interactions in Response to Cardiac Injury

A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. The electrophysiological consequences of this phenotypic switch remain largely unknown. Determine whether fibroblast activation following cardiac injury results in a distinct electrophysiological phenotype that enhances fibroblast-myocyte interactions. Neonatal rat myocyte monolayers were treated with media (CM) conditioned by fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts. Fb and MI-Fb were also plated on top of myocyte monolayers at 3 densities. Cultures were optically mapped after CM treatment or fibroblast plating to obtain conduction velocity and action potential duration (APD(70)). Intercellular communication and connexin43 expression levels were assessed. Membrane properties of Fb and MI-Fb were evaluated using patch clamp techniques. MI-Fb CM treatment decreased conduction velocity (11.1%) compared to untreated myocyte cultures. APD(70) was reduced by MI-Fb CM treatment compared to homocellular myocyte culture (9.4%) and Fb CM treatment (6.4%). In heterocellular cultures, MI-Fb conduction velocities were different from Fb at all densities (+29.8%, -23.0%, and -16.7% at 200, 400, and 600 cells/mm(2), respectively). APD(70) was reduced (9.6%) in MI-Fb compared to Fb cultures at 200 cells/mm(2). MI-Fb had more hyperpolarized resting membrane potentials and increased outward current densities. Connexin43 was elevated (134%) in MI-Fb compared to Fb. Intercellular coupling evaluated with gap fluorescence recovery after photobleaching was higher between myocytes and MI-Fb compared to Fb. These data demonstrate cardiac injury results in significant electrophysiological changes that enhance fibroblast-myocyte interactions and could contribute to the greater incidence of arrhythmias observed in fibrotic hearts.

Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease

S100B, a calcium-binding protein of the EF-hand type, exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures and high level expression of S100B in transgenic mice hearts inhibit cardiac hypertrophy and the associated phenotype but augments myocyte apoptosis following myocardial infarction. By contrast, knocking out S100B, augments hypertrophy, decreases apoptosis and preserves cardiac function following myocardial infarction. Expression of S100B in aortic smooth muscle cells inhibits cell proliferation and the vascular response to adrenergic stimulation. S100B induces apoptosis by an extracellular mechanism via interaction with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular and extracellular roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular diseases.

Effect of essential oil of Syringa pinnatifolia Hemsl. var. alashanensis on ischemia of myocardium, hypoxia and platelet aggregation

Aim of the study To investigate the cardioprotective potential of Syringa pinnatifolia Hems1. var. alashanensis essential oil (SPEO) against experimental acute myocardial ischemia (AMI), hydrogen peroxide (H 2O 2)-induced myocyte injury and activities against hypoxia and platelet aggregation. Materials and methods Wistar rats, Kunming mice and primary cultured rat neonatal myocytes were used in this study. AMI in rats was induced by ligation of the left anterior descending (LAD) coronary artery, and deviation of ST-segment, as well as changes of myocardial enzyme activities were observed. Hypoxia test in Kunming mice was performed to evaluate the effect of SPEO against hypoxia. The protective effect of SPEO on H 2O 2-induced cell injury was evaluated in terms of cell viability assay. The in vitro effect of SPEO against platelet aggregation was studied using adenosine 5′-diphosphate (ADP) as agonist. Results Administration of SPEO reduced deviation of ST-segment, decreased the levels of lactate dehydrogenase (LDH), creatine kinase (CK) and Troponin T (TnT) while increased the activities of superoxide dismutase (SOD). The protective role of SPEO was further confirmed by histopathological examination. In the hypoxia test, both 8 and 32 mg/kg of SPEO could prolong survival time of mice under hypoxia condition. At the meantime SPEO showed remarkable protective effect on cultured rat myocyte death induced by H 2O 2. SPEO also inhibited ADP-induced rat platelet aggregation by 47.4%, 37.0% and 32.9% at the dose of 5, 2.5 and 1.25 μg/mL, respectively. Conclusions These results suggest that SPEO possessing activities against hypoxia, oxidative stress and platelet aggregation has a significant protective effect against experimental myocardial ischemia.

Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor Fn14 during cardiac remodelling in rats

Accumulating evidence supports the concept that proinflammatory cytokines play an essential role in the failing heart. We examined the concomitant tumour necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 expression in myocytes in vitro as well as in vivo in cardiac remodelling. We assessed TWEAK and its receptor Fn14 expression in response to angiotensin (Ang) II, myocardial infarction (MI) as well as to local adenovirus-mediated p38 gene transfer in vivo. The effect of various hypertrophic factors and mechanical stretch was studied in neonatal rat ventricular myocyte cell culture. Ang II increased Fn14 levels from 6 h to 2 weeks, the greatest increase in mRNA levels being observed at 6 h (6.3-fold, P < 0.001) and protein levels at 12 h (4.9-fold, P < 0.01). TWEAK mRNA and protein levels remained almost unchanged during Ang II infusion. Likewise, a rapid and sustained elevation of Fn14 mRNA and protein levels in the left ventricle was observed after experimental MI. Moreover, local p38 gene transfer increased Fn14 mRNA and protein but not TWEAK levels. Fn14 immunoreactive cells were mainly proliferating non-myocytes in the inflammation area while TWEAK immunoreactivity localized to cardiomyocytes and endothelial cells of the coronary arteries. Hypertrophic agonists and lipopolysaccharide increased Fn14 but not TWEAK gene expression in neonatal rat myocytes, while mechanical stretch upregulated Fn14 and downregulated TWEAK gene expression. In conclusion, the cardiac TWEAK/Fn14 pathway is modified in response to myocardial injury, inflammation and pressure overload. Furthermore, our findings underscore the importance of Fn14 as a mediator of TWEAK/Fn14 signalling in the heart and a potential target for therapeutic interventions.

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca 2+ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca 2+ imaging, we found that the depletion of ER/SR Ca 2+ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca 2+ ([Ca 2+] i), followed by sustained increase depending on extracellular Ca 2+. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na +/Ca 2+ exchanger inhibitors, inhibited [Ca 2+] i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl 3) or by an increased extracellular Ca 2+([Ca 2+] o) increased the concentration of intracelluar Ca 2+, whereas, the sustained elevation of [Ca 2+] i was reduced in the presence of SKF96365. Similarly, the duration of [Ca 2+] i increase was also shortened in the absence of extracellular Ca 2+. Western blot analysis showed that GdCl 3 increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl 3. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca 2+-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

The p75 neurotrophin receptor, semaphorins, and sympathetic traffic in the heart

the sympathetic and parasympathetic branches of the autonomic nervous system control cardiac function through the coordinated activity generated by neurons within the intracardiac autonomic ganglia and central nervous system (3). Activation of the sympathetic efferent projections to the heart increases heart rate, modifies the pattern and conduction velocity of the impulse through the heart, and augments both atrial and ventricular contractility while hastening myocardial relaxation. In the vasculature, the sympathetic nervous system (SNS) reduces venous capacitance and constricts resistance vessels, increasing blood pressure. Conversely, the parasympathetic nervous system (PSNS) decreases heart rate and cardiac impulse conduction. More recent evidence has suggested that the PSNS may also affect ventricular function, indirectly by countering β-adrenergic action or directly via inhibition of L-type Ca2+ channels (34). A transient imbalance between these two branches can lead to increased heart rate and blood pressure when cardiac output is increased. When subnormal cardiac PSNS activity concomitant with sympathetic hyperactivation persists, such as after myocardial infarction, this disequilibrium may increase the risk for cardiac adverse events, including arrhythmias, hypertension, cardiac hypertrophy, and, ultimately, heart failure. This condition is called “autonomic neural remodeling” and can greatly contribute to the induction of ventricular fibrillation, thus increasing the patient's susceptibility to sudden cardiac death (41, 47). It is plausible that neurotrophic factors, target organ-secreted proteins essential for the development and differentiation of neurons, are involved in the remarkable neuroplasticity exhibited by the SNS in the adult life, including autonomic remodeling after infarction and/or in the onset and progression of congestive heart failure (17, 30, 32).

S100B: a multifunctional role in cardiovascular pathophysiology

S100B, a calcium-binding protein of the EF-hand type exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures, and high level expression of S100B in transgenic mice hearts and aortic smooth muscle cells inhibit cardiac hypertrophy and the associated phenotype, arterial smooth muscle proliferation, respectively, but demonstrate increased apoptosis following α(1)-adrenergic stimulation or myocardial infarction. Knocking out S100B, augmented hypertrophy, decreased apoptosis and preserved cardiac function following myocardial infarction. S100B induces apoptosis by an extracellular mechanism by interacting with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular, and extracellular, roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular disease.

Resveratrol inhibits right ventricular hypertrophy induced by monocrotaline in rats

1. Resveratrol (RSV), a polyphenol in red wine, exhibits cardioprotective effects in vitro, such as inhibition of angiotensin II- or phenylephrine-induced cardiomyocyte hypertrophy in rat neonatal myocyte cultures and suppression of cardiac fibroblast proliferation. The aim of the present study was to investigate the protective effects of RSV against monocrotaline (MCT)-induced right ventricular (RV) hypertrophy in rats. 2. Male Sprague-Dawley rats were given a single injection of MCT (50 mg/kg, s.c.) and were then treated with either vehicle (normal saline) or RSV (10 and 30 mg/kg, i.g., twice daily) for 21 days. A separate group of control rats were not injected with MCT and were treated with normal saline for 21 days. At the end of the treatment period, all rats were subjected to echocardiography and haemodynamic measurements. In addition, after rats had been killed, the hearts were subjected to histopathological, untrastructural and immunohistochemical analyses. 3. In vehicle-treated rats, MCT injection resulted in 33% mortality, whereas mortality in RSV-treated MCT-injected rats was 0%. In vehicle-treated rats, MCT increased RV free wall thickness and RV systolic pressure and decreased pulmonary arterial acceleration time at the end of the experimental period. These dynamic changes were ameliorated by RSV in a dose-dependent manner. Histologically, MCT injection resulted in RV hypertrophy, swollen mitochrondria and cardiomyocyte apoptosis; all these morphological changes were dose-dependently improved in rats treated with RSV. 4. In conclusion, RSV inhibits the RV hypertrophy induced by MCT in rats and this effect is mediated by both a direct effect of RSV on cardiomyocytes and an indirect effect mediated via a reduction in pulmonary hypertension.

Efficient Biolistic Transfection of Fresh Adult Cardiac Myocytes with a Tagged Kv1.5 Channel

Modulation of ion channel trafficking is a potent means by which a cardiomyocyte can regulate its excitability. Much has been learned about the roles of motifs within K+ channels that affect their trafficking to the cell surface. However, by necessity, previous studies have relied on model expression systems, because the transfection of adult cardiomyocytes has, to date, proved intractable. Rat neonatal myocytes can be transfected but the currents expressed in these cells are quite different from those of adult cardiomyocytes. Viral transduction systems are effective in adult cells but require sophisticated containment facilities and prolonged culture of the myocytes, during which time substantial dedifferentiation generally occurs.We have developed a new method that, for the first time, allows the ready and convenient transfection of acutely isolated adult rat cardiac myocytes. Using a low pressure adaptation of a Bio-Rad Helios gene gun procedure, we have achieved efficient transfection of rat ventricular myocytes bombarded within two hours of myocyte isolation with gold particles coated with pcDNA3 constructs encoding tagged Kv1.5 constructs. Expression is rapid, robust, and detectable less than 24 hours post-transfection in myocytes retaining both current profiles and gross morphology comparable to freshly isolated cells. Using this system, we unequivocally demonstrate that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes and that it is expressed at the surface of that structure. We further demonstrate that Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disk per se, was generally unaffected. Thus, this new transfection method is an effective tool for the study of cardiac ion channel expression and targeting in a physiologically relevant system.

Mechanisms of Myocyte Cytotoxicity Induced by the Multikinase Inhibitor Sorafenib

The use of the anticancer multikinase inhibitor sorafenib is associated with cardiac ischemia or infarction and an increase in hypertension. We investigated various mechanisms that might be responsible for its cardiotoxicity in a neonatal rat myocyte model. As measured by lactate dehydrogenase release, sorafenib treatment of myocytes caused dose-dependent damage at therapeutically relevant concentrations. It had been hypothesized that inhibition of RAF1 and BRAF kinases may be responsible for sorafenib induced cardiotoxicity. However, because sorafenib treatment did not inhibit phosphorylation of ERK (extracellular signal-regulated kinase), it was concluded that sorafenib did not exert its damaging effects through RAF inhibition of the RAF/MEK/ERK kinase cascade. The clinically approved doxorubicin cardioprotective agent dexrazoxane did not protect myocytes from damage. At lower sorafenib concentrations, at least, these results are consistent with sorafenib not being able to induce significant oxidative damage. In conclusion, given the extreme lack of kinase selectivity that sorafenib exhibits, it is likely that inhibition of kinases other than RAF, or combinations of kinases, contributes to the cardiotoxic effects of sorafenib.

S100B Interaction With the Receptor for Advanced Glycation End Products (RAGE)

Post-myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis. This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model. In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations > or = 50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis. These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

Silencing the myotrophin gene by RNA interference leads to the regression of cardiac hypertrophy

Myotrophin-induced activation of NF-kappaB has been shown to be associated with cardiac hypertrophy (CH) that progresses to heart failure (HF). In the present study, we examined the cause-and-effect relationship between myotrophin and NF-kappaB activation using small hairpin RNA (shRNA) against myotrophin both in vitro (using neonatal rat myocytes) and in vivo [using myotrophin transgenic (Myo-Tg) mice, which overexpress myotrophin in the heart, develop CH, and gradually progress to HF]. Among several lentiviral vectors expressing myotrophin shRNAs, L-sh-109 showed the best silencing effect at both the mRNA (155.3 +/- 5.9 vs. 32.5 +/- 5.5, P < 0.001) and protein levels associated with a significant reduction of atrial natriuretic factor (ANF) and NF-kappaB. In vivo, when L-sh-109 was delivered directly into the hearts of 10-wk-old Myo-Tg mice, we observed a significant regression of cardiac mass (8.0 vs. 5.7 mg/g, P < 0.001) and myotrophin gene expression (54.5% over untreated Myo-Tg mice, P < 0.001) associated with a reduction in ANF and NF-kappaB signaling components. Our data suggest that using RNA interference to silence the myotrophin gene prevents NF-kappaB activation, associated with an attenuation of CH. This strategy could be an excellent therapeutic means for the treatment of CH and HF.