Embryonic Myocytes Research Articles

Manipulation of mitochondrial pathways in the neonatal period is associated with enhanced cardiac function

Purpose: Current treatments for neonatal cardiomyopathy and heart failure are non-specific; the use of vasoactive medications and mechanical supports predominate. Recent basic science studies show that embryonic myocytes have less active mitochondria and an immature cellular structure. In addition, the neonatal heart appears to be more closely related in structure to the embryonic/fetal heart with progressive differentiation into its mature form over time. Mitochondria appear to regulate these changes during the neonatal transition. In addition, there appears to be a window in the first week of life, where the neonatal …

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte’s molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

Neonatal Permeability Transition Pore Closure is Associated with Increased Cardiac Function

Introduction: Recent studies have shown that embryonic myocytes have less active mitochondria and immature cellular structure. The neonatal heart is also closer in structure and function to the fetal heart, but progressively differentiates into its mature form with time. Mitochondria appear to regulate these changes during the neonatal transition. The closure of the mitochondrial permeability transition pore (PTP), by pharmacologic or genetic inhibition of Cyclophilin D (CyPD), is associated with differentiation in cultured neonatal myocytes. We evaluated CyPD inhibition's effect on in vivo cardiac function. We hypothesized that closure of the mPTP in the neonatal mouse heart will increase cardiac function by enhancing myocyte differentiation and maturation without longer-term deleterious effects. Methods: One-day-old WT or CyPD null mice were treated with daily intraperitoneal injections of vehicle (inert), Cyclosporin A (CsA), or NIM811 for five days. At six days old and weanling, the mice underwent echocardiography to evaluate ejection fraction (EF), a measure of cardiac function. Hearts were then harvested for further biochemical analysis.Results: WT mice hearts treated with CsA or NIM811, as well as CyPD nulls, had significantly higher EF at six-days-old (P<0.0001) compared to WT untreated or vehicle-treated mice. At weanling, there was no significant difference in EF across all groups and all survived to this time point. Work regarding assays of electron transport chain function and structure remains ongoing. Conclusion: Our results demonstrate that PTP closure in the neonatal mouse heart is associated with increased EF, as compared to vehicle treated mice. Our most recent data suggests that there is no significant difference in EF at weanling across all groups. This suggests that PTP closure enhances the neonatal transition to a more differentiated myocardium leading to an early increase in ejection fraction usually observed in mature animals.

Effects of bepridil on stretch-activated BKca channels and stretch-induced extrasystoles in isolated chick hearts.

Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SAC(NS)) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (P(O)). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd(3+)). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd(3+), which blocks both SAKca and SAC(NS), on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possible also blocks SAC(NS), and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).

Preventing permeability transition pore opening increases mitochondrial maturation, myocyte differentiation and cardiac function in the neonatal mouse heart

In embryonic myocytes, closure of the mitochondrial permeability transition pore (PTP) drives mitochondrial maturation and cardiac myocyte differentiation. Since neonatal cardiac myocytes remain relatively immature, we hypothesized that inducing PTP closure at this age, by inhibiting the PTP regulator, cyclophilin D (CyPD), genetically or with Cyclosporin A (CsA) and NIM811, would increase cardiac function by increasing mitochondrial maturation and myocyte differentiation. Cultured neonatal myocytes or neonatal mice were treated for 5 d with vehicle, CsA or NIM811. Mitochondrial function and structure were measured in vitro. Myocyte differentiation was assessed by immunolabeling for contractile proteins. Cardiac function was determined using echocardiography. The probability of PTP opening was high in WT neonatal myocytes. Treatment with CsA or NIM811 in vitro increased mitochondrial structural complexity and membrane potential, decreased reactive oxygen species levels, and increased myocyte differentiation. WT mice treated with either CsA or NIM811 in vivo for the first 5 d of life had higher ejection fractions. Deleting CyPD had similar effects as CsA and NIM811 on all parameters. It may be feasible to inhibit the PTP using available drugs to increase mitochondrial maturation, myocyte differentiation, and cardiac function in neonates.

Physiological role of FGF signaling in growth and remodeling of developing cardiovascular system.

Fibroblast growth factor (FGF) signaling plays an important role during embryonic induction and patterning, as well as in modulating proliferative and hypertrophic growth in fetal and adult organs. Hemodynamically induced stretching is a powerful physiological stimulus for embryonic myocyte proliferation. The aim of this study was to assess the effect of FGF2 signaling on growth and vascularization of chick embryonic ventricular wall and its involvement in transmission of mechanical stretch-induced signaling to myocyte growth in vivo. Myocyte proliferation was significantly higher at the 48 h sampling interval in pressure-overloaded hearts. Neither Western blotting, nor immunohistochemistry performed on serial paraffin sections revealed any changes in the amount of myocardial FGF2 at that time point. ELISA showed a significant increase of FGF2 in the serum. Increased amount of FGF2 mRNA in the heart was confirmed by real time PCR. Blocking of FGF signaling by SU5402 led to decreased myocyte proliferation, hemorrhages in the areas of developing vasculature in epicardium and digit tips. FGF2 synthesis is increased in embryonic ventricular cardiomyocytes in response to increased stretch due to pressure overload. Inhibition of FGF signaling impacts also vasculogenesis, pointing to partial functional redundancy in paracrine control of cell proliferation in the developing heart.

Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits – poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) – as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.

GW24-e3922 Role of intracellular calcium handling in neighboring myocytes in regulating adult myocyte dedifferentiation/re-differentiation

ObjectivesAdult myocyte dedifferentiation/re-differentiation (AMDR) occurs during both acute and chronic cardiac diseases. AMDR-based regenerative circuits have been considered as conduits through which this mechanism achieves cardiac repair. However, the mechanism...

Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality

Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] KATP channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from KATP overexpression, wild type (WT) and KATP overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in KATP overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5days post conception (dpc). KATP currents were detectable in both WT and KATP-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5dpc). In contrast to adult cardiomyocytes, WT and KATP-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous KATP current, implying that these channels may be open and active under physiological conditions. At 9.5dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, KATP channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the KATP channel may have an important physiological role during early cardiac development.

Tbx3: a new trick for an 'old' myocyte?

This editorial refers to ‘T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells’ by M.L. Bakker et al ., doi:10.1093/cvr/cvs120. Tbx3: a new trick for an ‘old’ myocyte? Well, not quite … Tbx3 is a T-box transcription factor that is expressed in the pacemaking and conduction system myocytes beginning during early embryonic stages of cardiac development.1 When expressed in embryonic atrial chamber myocytes, ectopic induction of spontaneous pacemaking activity in atrial myocardium was observed in vivo , thereby suggesting that Tbx3 can ‘impose’ pacemaking capability upon otherwise unsuspecting working myocytes.2 Bakker et al. 3 test the hypothesis that Tbx3 would also be able to induce pacemaker activity in mature adult cardiac myocytes. Like the proverbial ‘old dog’, however, it appears that it is also difficult to teach old myocytes this new trick. Bakker et al .3 show that while Tbx3 does repress certain genes typically excluded from pacemaking centres such as those encoding the gap junction proteins connexin 40 (Cx40) and connexin 43 (Cx43) in adult mouse atrial myocytes, it failed to induce the pacemaker channel protein encoded by the hyperpolarization-activated cyclic nucleotide-gated 4 ( Hcn4 ) gene. Consequently, no spontaneous sinusoidal action potentials characteristic of those typically observed in sinoatrial node pacemaking myocytes …

FGF signaling is involved in physiological adaptation to pressure overload in developing heart

Fibroblast growth factors (FGFs) play an important role during embryonic induction, growth and patterning. Exogenous FGF2 as well as pressure loading were shown to induce embryonic myocyte proliferation. Here we tested whether FGF2 signaling is involved in transmission of pressure overload to increased myocyte proliferation in vivo.Pressure overload was induced by conotruncus constriction in Stage 21 chick embryos. Anti‐bromodeoxyuridine labeling was used to assess proliferation at 24, 48 and 72 h. Western blotting and RT‐PCR to measure FGF2 protein and mRNA levels was performed on ventricular extracts at 48 h interval. Blood was collected at 48 h to assess the amount of FGF2 in serum by ELISA. Proliferation of ventricular myocytes was increased significantly at 48 h. Neither western blotting, nor immunohistochemistry revealed any changes in the amount of myocardial FGF2. ELISA showed a 93% increase of FGF2 in the serum. Increased amount of FGF2 mRNA in ventricular myocardium was confirmed by real time PCR.We conclude that FGF2 synthesis is increased in embryonic ventricular cardiomyocytes in response to increased stretch due to pressure overload. Increased stretch causes its release into serum, causing it to act in endocrine, rather then paracrine manner.Supported by Ministry of Education VZ 0021620806, ASCR AV0Z50450515 and AV0Z50110509 and GACR P302/11/1308.Grant Funding Source : P302/11/1308

Norepinephrine causes epigenetic repression of PKCε gene in rodent hearts by activating Nox1‐dependent reactive oxygen species production

Heart disease is the leading cause of death in the United States. Recent studies demonstrate that fetal programming of PKCε gene repression results in ischemia-sensitive phenotype in the heart. The present study tests the hypothesis that increased norepinephrine causes epigenetic repression of PKCε gene in the heart via Nox1-dependent reactive oxygen species (ROS) production. Prolonged norepinephrine treatment increased ROS production in fetal rat hearts and embryonic ventricular myocyte H9c2 cells via a selective increase in Nox1 expression. Norepinephrine-induced ROS resulted in an increase in PKCε promoter methylation at Egr-1 and Sp-1 binding sites, leading to PKCε gene repression. N-acetylcysteine, diphenyleneiodonium, and apocynin blocked norepinephrine-induced ROS production and the promoter methylation, and also restored PKCε mRNA and protein to control levels in vivo in fetal hearts and in vitro in embryonic myocyte cells. Accordingly, norepinephrine-induced ROS production, promoter methylation, and PKCε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. These findings provide evidence of a novel interaction between elevated norepinephrine and epigenetic repression of PKCε gene in the heart mediated by Nox1-dependent oxidative stress and suggest new insights of molecular mechanisms linking the heightened sympathetic activity to aberrant cardioprotection and increased ischemic vulnerability in the heart.

Abstract P005: Direct Differentiation of Atrial and Ventricular Myocytes from Human Embryonic Stem Cells

Although cell transplantation studies have suggested promising therapeutic potentials for myocardial infarction, the leading cause of death worldwide, the incapability to obtain relatively homogeneous ventricular myocytes for transplantation is one major obstacle to the development of clinical therapies for myocardial repair1. Human embryonic stem cell (hESC) is a promising source of cardiomyocytes. Here we show that both Noggin and pan-retinoic acid receptor antagonist BMS-1894532 (RAi) significantly increase the cardiac differentiation efficiency of hESCs. Investigating retinoid function by comparing Noggin+RAi-treated cultures with Noggin+RA-treated cultures, we found that with 64±0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (AP); while, with 50±1.76% cardiac differentiation efficiency, 94% of those in Noggin+RA-treated cultures had embryonic atrial-like APs. These findings demonstrating that relatively homogeneous embryonic atrial and ventricular myocyte populations can be efficiently derived from hESCs by specifically influencing signaling cascades.

Development of the Pacemaker Tissues of the Heart

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues.

Fibroblast Growth Factor-2 regulates proliferation of cardiac myocytes in normal and hypoplastic left ventricles in the developing chick

The developing heart increases its mass predominantly by increasing the number of contained cells through proliferation. We hypothesized that addition of fibroblast growth factor-2, a factor previously shown to stimulate division of the embryonic myocytes, to the left ventricular myocardium in an experimental model of left heart hypoplasia created in the chicken would attenuate phenotypic severity by increasing cellular proliferation. We have established an effective mode of delivery of fibroblast growth factor-2 to the chick embryonic left ventricular myocardium by using adenovirus vectors, which was more efficient and better tolerated than direct injection of recombinant fibroblast growth factor-2 protein. Injection of control adenovirus expressing green fluorescent protein did not result in significant alterations in myocytic proliferation or cell death compared with intact, uninjected, controls. Co-injection of adenoviruses expressing green fluorescent protein and fibroblast growth factor-2 was used for verification of positive injection, and induction of proliferation, respectively. Treatment of both normal and hypoplastic left ventricles with fibroblast growth factor-2 expressing adenovirus resulted in to 2 to 3-fold overexpression of fibroblast growth factor-2, as verified by immunostaining. An increase by 45% in myocytic proliferation was observed following injection of normal hearts, and an increase of 39% was observed in hypoplastic hearts. There was a significant increase in anti-myosin immunostaining in the hypoplastic, but not the normal hearts. We have shown, therefore, that expression of exogenous fibroblast growth factor-2 in the late embryonic heart can exert direct effects on cardiac myocytes, inducing both their proliferation and differentiation. These data suggest potential for a novel therapeutic option in selected cases of congenital cardiac disease, such as hypoplastic left heart syndrome.

Myotonic dystrophy protein kinase is expressed in embryonic myocytes and is required for myotube formation

Myotonic dystrophy (DM1) is a multi-systemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). The primary pathophysiology of DM1 is thought to result from RNA transport and processing defects. The function of DMPK in development or any potential role in DM1 remains unknown. Here we report a novel role for DMPK in myogenesis. We have discovered a specific expression pattern of DMPK in mouse and chick embryonic development. DMPK is expressed in postmitotic cardiac and skeletal myocytes and developmental signaling centers. During cardiac myocyte maturation, DMPK migrates from perinuclear to cellular membrane localization. Manipulating DMPK levels in cultured cardiac and skeletal myocytes has revealed a key role for DMPK in myocyte differentiation. Overexpression of DMPK induces cell rounding and apoptosis in myocytes. In addition, DMPK is necessary for myogenin expression in differentiating C2C12 myoblasts.

Embryonic myocyte‐derived factors regulate early coronary vessel development

Many heart problems are associated with defects and disease of the coronary vessels; however coronary vessel development is not well understood. Coronary vessels originate from epicardial cells which come from the proepicardium (PE). At E9.0 in mice, PE cells migrate to and spread over the heart to form the epicardium. A subset of epicardial cells undergoes an epithelial to mesenchymal transformation (EMT) and migrates into the myocardium. These epicardial derived cells (EDC) differentiate into epithelial and smooth muscle cells of the coronary vessels. Signals required for this process are speculated to originate from the myocardium and endocardium, but there is no direct evidence for this activity. We have developed a novel in vitro system to directly address the question of “where do the signals required for early coronary vessel development originate?” In this system we co‐culture primary epicardial monolayers with embryonic myocytes. We detect an increase in epicardial EMT during co‐culture conditions compared to naïve epicardial cultures. We have identified several growth factors, including VEGF and TGF‐β2, which are produced by embryonic myocytes in vitro. Thus, our system allows for the identification of mediators responsible for epicardial EMT and, ultimately, EDC differentiation into the coronary vasculature. Supported by HL077493 and T32 HL07249.

Cardiac expression patterns of endothelin‐converting enzyme (ECE) suggest a role of endothelin signaling in conduction system development

Conversion of big-endothelin (ET) into biologically active ET is performed proteolytically by endothelin-converting enzyme (ECE). ET has ability to induce differentiation of embryonic myocytes into a Purkinje fiber-like phenotype in vitro and in vivo. We have generated an antibody against chick ECE to correlate its cardiac expression patterns reported previously at mRNA level. On Western blot, the antibody recognized a single band at ~70 kD molecular mass. Immunohistochemically the protein expression was more widespread compared to RNA, being present in the endothelium, as well as in mesenchymal cells and myocytes, and enriched in the ventricular trabeculae and nascent ventricular conduction system. At later fetal and postnatal stages, the expression was more restricted to the coronary endothelium and periarterial Purkinje fibers, partially overlapping with connexin40 expression. The myocardial expression was significantly decreased in experimental left ventricular hypoplasia, correlating with conduction deficiencies reported in this model previously. Inhibition of ET signaling by ET receptor antagonist bosentan resulted in delay of bundle branches maturation. Together, ECE expression patterns are consistent with the role of ET signaling in conduction system induction during avian cardiac development. Supported by NIH HL56728, HD 39946, RR 16434; MECR VZ 206100–3, and ASCR AVOZ50450515.

Ca2+ transient‐dependent changes in embryonic myocyte proteins during myofibrillogenesis: a proteomic analysis

Spontaneous intracellular calcium (Ca2+) signals, called transients, play a critical role during skeletal muscle myofibrillogenesis. In embryonic skeletal muscle, several classes of Ca2+ transients occur both in vivo and in culture, and blocking their production prevents de novo sarcomere assembly. Since Ca2+ signaling is paramount to a variety of cell signaling events, it is expected that perturbing these events would effect protein expression and/or posttranslational modification. We implemented a proteomics screen to assess global protein differences. We are using two-dimensional (2D) electrophoresis to study both the absolute protein amount as well as any modification that would affect pI. Protein lysates from whole embryos were optimized and enriched for myofibrillar proteins. Several protein spots showing a presumptive increase in Ca2+ dependent phosphorylation were identified by MS/MS. Other MS identified proteins showed marked decrease in sarcomeric incorporation. For example, the fast skeletal regulatory light chain (MLC2f) showed a decrease in both sarcomeric incorporation and phosphorylation when Ca2+ transients were inhibited. In order to assess whether MLC2f expression and/or phosphorylation are necessary for myofibrillogenesis, MLC2f expression was knocked down using morpholino technology. Embryos were completely paralyzed but otherwise appeared normal. MLC2f knockdown resulted in complete inhibition of sarcomeric myosin organization. We are currently creating MLC2f constructs for rescue and sufficiency experiments using both phosphodominant and phosphonegative site-directed mutagenesis.

NRSF regulates the developmental and hypertrophic changes of HCN4 transcription in rat cardiac myocytes

The HCN4 channel shows differential expression patterns during the embryonic development and hypertrophy of hearts. Briefly, HCN4 expression is maximally activated in embryonic hearts and quickly diminishes after birth. However, it is reactivated during cardiac hypertrophy. The sequence analysis of HCN4 gene revealed the presence of a conserved NRSE motif, which is known to bind the transcriptional factor neuron-restrictive silencing factor (NRSF). A promoter analysis of HCN4 with rat cardiac myocytes identified the region inducing a basal transcriptional activity. This region drove a high activity in embryonic myocytes, but not in neonatal myocytes treated with hypertrophic agents. After confirming that NRSF protein binds to the NRSE, HCN4 promoter activities modified by NRSE were evaluated. With wild-type NRSE, the promoter activity correlated well with the developmental and hypertrophic changes of HCN4 expression, whereas mutant NRSE constructs failed. We conclude that the NRSE-NRSF system was implicated in HCN4 expression in cardiac myocytes.