Neonatal Myocytes Research Articles

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.

Minocycline protects against myocardial ischemia and reperfusion injury by inhibiting high mobility group box 1 protein in rats

Minocycline has been shown to protect against myocardial ischemia and reperfusion injury. However, the mechanism remains unclear. This study was to investigate the role of high mobility group box 1 protein (HMGB1) in the cardioprotection of minocycline during myocardial ischemia and reperfusion in rats. Anesthetized male rats were once treated with minocycline (45 mg/kg, i.p.) 1 h before ischemia, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. The lactate dehydrogenase (LDH), creatine kinase (CK) and infarct size were measured and the myocardial tissue apoptosis was assessed by TUNNEL assay. Neonatal rat ventricular myocytes were prepared and then cultured with recombinant HMGB1. Cell apoptosis was measured using an annexin V-FITC apoptosis detection kit. HMGB1 expression was assessed by immunoblotting. After 4 h of reperfusion, minocycline could significantly decrease the infarct size, myocardium apoptosis and the levels of LDH and CK (all P < 0.05). Meanwhile, minocycline could also significantly inhibit the HMGB1 expression during myocardial ischemia and reperfusion compared to that in ischemia and reperfusion group ( P < 0.05). In vitro, HMGB1 could significantly decrease the cell viability and promote the apoptosis of neonatal myocytes in a dose-dependent manner. The present study suggested that minocycline could protect against myocardial ischemia and reperfusion injury by inhibiting HMGB1 expression.

The bHLH transcription factor CHF1/Hey2 regulates susceptibility to apoptosis and heart failure after pressure overload

Cardiac hypertrophy is a common response to hemodynamic stress in the heart and can progress to heart failure. To investigate whether the transcription factor cardiovascular basic helix-loop-helix factor 1/hairy/enhancer of split related with YRPW motif 2 (CHF1/Hey2) influences the development of cardiac hypertrophy and progression to heart failure under conditions of pressure overload, we performed aortic constriction on 12-wk-old male wild-type (WT) and heterozygous (HET) mice globally underexpressing CHF1/Hey2. After aortic banding, WT and HET mice showed increased cardiac hypertrophy as measured by gravimetric analysis, as expected. CHF1/Hey2 HET mice, however, demonstrated a greater increase in the ventricular weight-to-body weight ratio compared with WT mice (P < 0.05). Echocardiographic measurements showed a significantly decreased ejection fraction compared with WT mice (P < 0.05). Histological examination of Masson trichrome-stained heart tissue demonstrated extensive fibrosis in HET mice compared with WT mice. TUNEL staining demonstrated increased apoptosis in HET hearts (P < 0.05). Exposure of cultured neonatal myocytes from WT and HET mice to H(2)O(2) and tunicamycin, known inducers of apoptosis that work through different mechanisms, demonstrated significantly increased apoptosis in HET cells compared with WT cells (P < 0.05). Expression of Bid, a downstream activator of the mitochondrial death pathway, was expressed in HET hearts at increased levels after aortic banding. Expression of GATA4, a transcriptional activator of cardiac hypertrophy, was also increased in HET hearts, as was phosphorylation of GATA4 at Ser(105). Our findings demonstrate that CHF1/Hey2 expression levels influence hypertrophy and the progression to heart failure in response to pressure overload through modulation of apoptosis and GATA4 activity.

Down‐regulation of MnSOD via Sirt1/FoxO3a complex increase oxidative stress with cardiac overexpression of Type 5 Adenylyl Cyclase

We examined the stress of chronic (7 days) isoproterenol (ISO) stimulation 60mg/kg/day in 3–5 month AC5 Tg mice and WT littermates. Over 30% Tg mice died after day 3 while none of the WT mice died (n=12/group). Oxidative stress induced by chronic ISO stimulation was detected by 8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) staining and AC5 Tg hearts showed 27% more positive staining than WT. To further examine whether AC5 Tg mice are less tolerant to oxidative stress, we injected paraquat (PQ), an oxidative stress inducer, and again more positive 8‐OHdG staining was observed in AC5 Tg mice hearts than AC5 KO mice, while the anti‐oxidant MnSOD protein was 40% lower in AC5 Tg than WT. We further hypothesized that inhibition of AC5 activates Sirt1 and FoxO3a, key transcription regulators of MnSOD. Luciferase test showed 3 fold higher FoxO3a transcriptional activity in AC5 knockdown myocytes. Protein expression of MnSOD was 40% decreased in neonatal myocytes infected by both ad‐AC5‐shRNA and ad‐Sirt1‐shRNA compared with AC5 knockdown myocytes. These data suggest that AC5 inhibits the Sirt1/FoxO3a complex thus down‐regulating MnSOD expression, and consequently increasing oxidative stress.

TRPC channels are necessary mediators of pathologic cardiac hypertrophy

Pathologic hypertrophy of the heart is regulated through membrane-bound receptors and intracellular signaling pathways that function, in part, by altering Ca(2+) handling and Ca(2+)-dependent signaling effectors. Transient receptor potential canonical (TRPC) channels are important mediators of Ca(2+)-dependent signal transduction that can sense stretch or activation of membrane-bound receptors. Here we generated cardiac-specific transgenic mice that express dominant-negative (dn) TRPC3, dnTRPC6, or dnTRPC4 toward blocking the activity of the TRPC3/6/7 or TRPC1/4/5 subfamily of channels in the heart. Remarkably, all three dn transgenic strategies attenuated the cardiac hypertrophic response following either neuroendocrine agonist infusion or pressure-overload stimulation. dnTRPC transgenic mice also were partially protected from loss of cardiac functional performance following long-term pressure-overload stimulation. Importantly, adult myocytes isolated from hypertrophic WT hearts showed a unique Ca(2+) influx activity under store-depleted conditions that was not observed in myocytes from hypertrophied dnTRPC3, dnTRPC6, or dnTRPC4 hearts. Moreover, dnTRPC4 inhibited the activity of the TRPC3/6/7 subfamily in the heart, suggesting that these two subfamilies function in coordinated complexes. Mechanistically, inhibition of TRPC channels in transgenic mice or in cultured neonatal myocytes significantly reduced activity in the calcineurin-nuclear factor of activated T cells (NFAT), a known Ca(2+)-dependent hypertrophy-inducing pathway. Thus, TRPC channels are necessary mediators of pathologic cardiac hypertrophy, in part through a calcineurin-NFAT signaling pathway.

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.

Characterisation and regulation of E2F-6 and E2F-6b in the rat heart: a potential target for myocardial regeneration?

The E2F transcription factors are instrumental in regulating cell cycle progression and growth, including that in cardiomyocytes, which exit the cell cycle shortly after birth. E2F-6 has been demonstrated to act as a transcriptional repressor; however, its potential role in normal cardiomyocyte proliferation and hypertrophy has not previously been investigated. Here we report the isolation and characterisation of E2F-6 and E2F-6b in rat cardiomyocytes and consider its potential as a target for myocardial regeneration following injury. At the mRNA level, both rat E2F-6 and the alternatively spliced variant, E2F-6b, were expressed in E18 myocytes and levels were maintained throughout development into adulthood. Interestingly, E2F-6 protein expression was down-regulated during myocyte development suggesting that it is regulated post-transcriptionally in these cells. During myocyte hypertrophy, the mRNA expressions of E2F-6 and E2F-6b were not regulated whereas E2F-6 protein was up-regulated significantly. Indeed, E2F-6 protein expression levels closely parallel the developmental withdrawal of myocytes from the cell cycle and the subsequent reactivation of their cell cycle machinery during hypertrophic growth. Furthermore, depletion of E2F-6, using anti-sense technology, results in death of cultured neonatal myocytes. Taken together, abrogation of E2F-6 expression in neonatal cardiomyocytes leads to a significant decrease in their viability, consistent with the notion that E2F-6 might be required for maintaining normal myocyte growth.

Characterization and expression of a heart-selective alternatively spliced variant of αII-spectrin, cardi+, during development in the rat

Spectrin is a large, flexible protein that stabilizes membranes and organizes proteins and lipids into microdomains in intracellular organelles and at the plasma membrane. Alternative splicing occurs in spectrins, but it is not yet clear if these small variations in structure alter spectrin's functions. Three alternative splice sites have been identified previously for αII-spectrin. Here we describe a new alternative splice site, a 21-amino acid sequence in the 21st spectrin repeat that is only expressed in significant amounts in cardiac muscle (GenBank GQ502182). The insert, which we term αII-cardi+, results in an insertion within the high affinity nucleation site for binding of α-spectrins to β-spectrins. To assess the developmental regulation of the αII-cardi+ isoform, we used qRT-PCR and quantitative immunoblotting methods to measure the levels of this form and the αII-cardi− form in the cardiac muscles of rats, from embryonic day 16 (E16) through adulthood. The αII-cardi+ isoform constituted ∼26% of the total αII-spectrin in E16 hearts but decreased to ∼6% of the total after 3 weeks of age. We used long-range RT-PCR and Southern blot hybridization to examine possible linkage of the αII-cardi+ alternatively spliced sequence with alternatively spliced sequences of αII-spectrin that had been previously reported. We identified two new isoforms of αII-spectrin containing the cardi+ insert. These were named αIIΣ9 and αIIΣ10 in accordance with the spectrin naming conventions. In vitro studies of recombinant αII-spectrin polypeptides representing the two splice variants of αII-spectrin, αII-cardi+ and αII-cardi−, revealed that the αII-cardi+ subunit has lower affinity for the complementary site in repeats 1–4 of βII-spectrin, with a KD value of ∼1 nM, as measured by surface plasmon resonance (SPR). In addition, the αII-cardi+ form showed 1.8-fold lower levels of binding to its site on βII-spectrin than the αII-cardi− form, both by SPR and blot overlay. This suggests that the 21-amino acid insert prevented some of the αII-cardi+ form from interacting with βII-spectrin. Fusion proteins expressing the αII-cardi+ sequence within the two terminal spectrin repeats of αII-spectrin were insoluble in solution and aggregated in neonatal myocytes, consistent with the possibility that this insert removes a significant portion of the protein from the population that can bind β subunits. Neonatal rat cardiomyocytes infected with adenovirus encoding GFP-fusion proteins of repeats 18–21 of αII-spectrin with the cardi+ insert formed many new processes. These processes were only rarely seen in myocytes expressing the fusion protein lacking the insert or in controls expressing only GFP. Our results suggest that the embryonic mammalian heart expresses a significant amount of αII-spectrin with a reduced avidity for β-spectrin and the ability to promote myocyte growth.

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.

Cardiomyocyte cell cycle control and growth estimation in vivo—an analysis based on cardiomyocyte nuclei

Adult mammalian cardiomyocytes are traditionally viewed as being permanently withdrawn from the cell cycle. Whereas some groups have reported none, others have reported extensive mitosis in adult myocardium under steady-state conditions. Recently, a highly specific assay of 14C dating in humans has suggested a continuous generation of cardiomyocytes in the adult, albeit at a very low rate. Mice represent the most commonly used animal model for these studies, but their short lifespan makes them unsuitable for 14C studies. Herein, we investigate the cellular growth pattern for murine cardiomyocyte growth under steady-state conditions, addressed with new analytical and technical strategies, and we furthermore relate this to gene expression patterns. The observed levels of DNA synthesis in early life were associated with cardiomyocyte proliferation. Mitosis was prolonged into early life, longer than the most conservative previous estimates. DNA synthesis in neonatal life was attributable to bi-nucleation, therefore suggesting that cardiomyocytes withdraw from the cell cycle shortly after birth. No cell cycle activity was observed in adult cardiomyocytes and significant polyploidy was observed in cardiomyocyte nuclei. Gene analyses identified 32 genes whose expression was predicted to be particular to day 3-4 neonatal myocytes, compared with embryonic or adult cells. These cell cycle-associated genes are crucial to the understanding of the mechanisms of bi-nucleation and physiological cellular growth in the neonatal period.

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (Ito) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in Ito-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased Ito densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca2+ transients. Interestingly, blocking Ito with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca2+ transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca2+-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.

Developmental Changes of the Sarcoplasmic Reticulum

In ventricular myocytes, excitation-contraction coupling (ECC), or the process by which an action potential leads to myocyte contraction, is reliant upon external influx of calcium (Ca+2) via sarcolemmal voltage-gated L-type Ca+2 channels to trigger Ca+2 release through nearby ryanodine receptors located in the sarcoplasmic reticulum (SR) via the mechanism of Ca+2-induced Ca+2 release. This causes a global increase in intracellular in Ca+2 concentration ([Ca+2]i) that activates contractile filaments. The level of expression of proteins and cellular organelles involved in cardiac ECC changes during development. These differences arise from adult myocytes being larger, having smaller surface-to-volume ratio and a developed transverse (T)-tubule system that tightly couples to the junctional SR. To investigate ECC developmental changes, we developed an adenovirus encoding a tagged red fluorescent protein with a retention signal for the sarcoplasmic reticulum (tRFP-SR). This approach allowed us for the first time to track in real-time SR structure and [Ca+2]i dynamics in neonatal and adult ventricular myocytes. Using confocal and total internal reflection fluorescence (TIRF) microscopy, we found that the SR within neonatal and adult ventricular cardiomyocytes forms a vast network that spans virtually the entire cell. Interestingly, we found that the SR is a highly dynamic network with relatively rapid changes in morphology in neonatal myocytes. Calcium sparks were observed from these SR structures, suggesting they harbor functional ryanodine receptors. In contrast to neonatal myocytes, the SR of adult ventricular myocytes is highly stable. Our findings suggest, that the fidelity of the [Ca+2]i transient during ECC in neonatal myocytes is not simply the result of ryanodine receptors in a static SR network. Instead, our data indicate it is the result of the activation of numerous couplings that form and dissolve rapidly between the sarcoplasmic reticulum and plasma membrane.

Regulation of the Transient Outward Potassium Current Itof in Cardiac Hypertrophy by Sphingosine-1-Phosphate Signaling

The fast transient outward potassium current (Ito,f), which is carried by voltage-gated Kv4.2 and Kv4.3 potassium channels and auxiliary subunit KChIP2, plays a critical role in early repolarization of the cardiac action potential. Ito,f and its gene products are strongly down-regulated in cardiac hypertrophy and disease, leading to altered excitation-contraction coupling and electrical activity as well as hypertrophy. Despite the importance of Ito,f in normal and diseased hearts, the regulation of Ito,f remains poorly understood. Studies have shown that the biologically active sphingolipid, sphingosine-1-phosphate (S1P), induces cardiac hypertrophy. In addition, the inflammatory pro-hypertrophic cytokine TNF-alpha, which decreases Ito,f, activates sphingosine kinase 1, the highly regulated enzyme that produces S1P. Therefore, we investigated the role of TNF-alpha and S1P signaling in mediating the down-regulation of Ito,f. In cultured neonatal myocytes, the TNF-alpha inhibitor etanercept attenuated reductions in Ito,f current density that were caused by the hypertrophic agonist phenylephrine (PE). Inhibition of sphingosine kinases by dimethylsphingosine prevented reductions in Ito,f that were caused by PE. Furthermore, application of S1P reduced Ito,f current density and caused hypertrophy. To interrogate the down-stream events involved in TNF-alpha/S1P- induced reductions in Ito,f, we focused on NF-kappaB since it is one of TNF-alpha's principal down-stream effectors and a major player in cardiac hypertrophy. Inhibition of NF-kappaB signaling, via an adenoviral expression of an IkappaB-alpha dominant negative mutant, prevented Ito,f reductions caused by S1P, TNF-alpha and PE. Taken together, the data suggests a linear pathway whereby PE promotes the shedding of TNF-alpha, which activates sphingosine kinase and elevates S1P, leading to activation of NF-kappaB and down-regulation of Ito,f.We will test this model in future experiments by assessing the effects of cardiac disease on Ito,f in knockout mice lacking sphingosine kinase and TNF-alpha.

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.

CHF1/Hey2 Promotes Physiological Hypertrophy in Response to Pressure Overload through Selective Repression and Activation of Specific Transcriptional Pathways

We have previously found that CHF1/Hey2 prevents the development of phenylephrine-induced cardiac hypertrophy. To determine the role of CHF1/Hey2 in pressure overload hypertrophy, we performed ascending aortic banding on wild-type and transgenic mice overexpressing CHF1/Hey2 in the myocardium. We found that both wild-type and transgenic mice developed increased ventricular weight to body weight ratios 1 week after aortic banding. Wild-type mice also developed decreased fractional shortening after 1 week when compared to preoperative echocardiograms and sham-operated controls. Transgenic mice, in comparison, demonstrated preserved fractional shortening. Histological examination of explanted heart tissue demonstrated extensive fibrosis in wild-type hearts, but minimal fibrosis in transgenic hearts. TUNEL staining demonstrated increased apoptosis in the wild-type hearts but not in the transgenic hearts. Exposure of cultured neonatal myocytes from wild-type and transgenic animals to hydrogen peroxide, a potent inducer of apoptosis, demonstrated increased apoptosis in the wild-type cells. Gene Set Analysis of microarray data from wild-type and transgenic hearts 1 week after banding revealed suppression and activation of multiple pathways involving apoptosis, cell signaling, and biosynthesis. These findings demonstrate that CHF1/Hey2 promotes physiological over pathological hypertrophy through suppression of apoptosis and regulation of multiple transcriptional pathways. These findings also suggest that CHF1/Hey2 and its downstream pathways provide a variety of targets for novel heart failure drug discovery, and that genetic polymorphisms in CHF1/Hey2 may affect susceptibility to hypertrophy and heart failure.

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.

The mAKAPβ scaffold regulates cardiac myocyte hypertrophy via recruitment of activated calcineurin

mAKAPβ is the scaffold for a multimolecular signaling complex in cardiac myocytes that is required for the induction of neonatal myocyte hypertrophy. We now show that the pro-hypertrophic phosphatase calcineurin binds directly to a single site on mAKAPβ that does not conform to any of the previously reported consensus binding sites. Calcineurin–mAKAPβ complex formation is increased in the presence of Ca 2+/calmodulin and in norepinephrine-stimulated primary cardiac myocytes. This binding is of functional significance because myocytes exhibit diminished norepinephrine-stimulated hypertrophy when expressing a mAKAPβ mutant incapable of binding calcineurin. In addition to calcineurin, the transcription factor NFATc3 also associates with the mAKAPβ scaffold in myocytes. Calcineurin bound to mAKAPβ can dephosphorylate NFATc3 in myocytes, and expression of mAKAPβ is required for NFAT transcriptional activity. Taken together, our results reveal the importance of regulated calcineurin binding to mAKAPβ for the induction of cardiac myocyte hypertrophy. Furthermore, these data illustrate how scaffold proteins organizing localized signaling complexes provide the molecular architecture for signal transduction networks regulating key cellular processes.

Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle.

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.

An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

Calcineurin Finds a New Partner in the L-Type Ca 2+ Channel

See related articles, pages 51–60 Calcineurin is a Ca2+/calmodulin-sensitive phosphatase that sits near the top of signaling pathways leading to pathological cardiac hypertrophy.1 Pathological stressors activate calcineurin, for which nuclear factor of activated T cells (NFAT) is a principal substrate. Dephosphorylation unmasks a nuclear localization signal on NFAT, which then translocates to the nucleus and, there, serves as part of a transcription factor complex able to initiate gene expression cascades that induce cardiac hypertrophy and subsequent heart failure. Determining how calcineurin is specifically activated in response to hypertrophic stressors by the same Ca2+ signal used for excitation–contraction coupling has been a major challenge. Triggered by Ca2+ influx through CaV1.2 voltage-gated Ca2+ channels at the sarcolemmal membrane, intracellular calcium briefly increases 10-fold with each heart beat to generate myocyte contraction. Almost as quickly, intracellular calcium falls to its baseline level, allowing myocyte relaxation and preparation for the next beat. There are 2 leading proposals for how Ca2+ could activate the calcineurin/NFAT pathway or function as a versatile regulator of multiple other signaling cascades in the face of the large Ca2+ oscillations driving each cycle of contraction and relaxation. One is that modulation of the amplitude and/or frequency of …