We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties.

HL-1 cells are currently the only cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype. Extensive characterization using microscopic, genetic, immunohistochemical, electrophysiological, and pharmacological techniques has demonstrated how similar HL-1 cells are to primary cardiomyocytes. In the few years that HL-1 cells have been available, they have been used in a variety of model systems designed to answer important questions regarding cardiac biology at the cellular and molecular levels. Whereas HL-1 cells have been used to study normal cardiomyocyte function with regard to signaling, electrical, metabolic, and transcriptional regulation, they have also been used to address pathological conditions such as hypoxia, hyperglycemia-hyperinsulinemia, apoptosis, and ischemia-reperfusion. The availability of an immortalized, contractile cardiac cell line has provided investigators with a tool for probing the intricacies of cardiomyocyte function. In this review, we describe the culture and characterization of HL-1 cardiomyocytes as well as various model systems that have been developed using these cells to gain a better understanding of cardiac biology at the cellular and molecular levels.

Abstract-Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. Key Words: nucleoside Ⅲ adenosine Ⅲ transport Ⅲ ischemia Ⅲ pH T he purine nucleoside adenosine is produced by the action of both endo-and ecto-nucleotidases on adenine nucleotides in the heart and plays key roles in the regulation of coronary blood flow and myocardial O 2 supply-demand balance. 1-4 For example, action of adenosine on A 2A receptors on vascular smooth muscle and endothelial cells causes coronary vasodilatation. 1,5 In contrast, the negative inotropic and dromotropic effects of adenosine on the heart are mediated primarily by A 1 receptors. 2 Similarly, the negative chromotropic effect of adenosine involves action of A 1 receptors in the sinoatrial (SA) node on the inwardly rectifying potassium channel current I K-Ado and the hyperpolarization-activated pacemaker current I f . 2,6 Endogenous adenosine, acting on mitochondrial K ATP channels via A 1 and A 3 receptors, also makes a major contribution to the phenomenon of ischemic preconditioning. 5,7 Extracellular adenosine concentrations in the heart are governed both by action of ecto-5Ј-nucleotidase on adenine nucleotides released from cells and by transporter-mediated flux of adenosine across cell membranes. 3,4 Although most adenosine production occurs intracellularly, under normoxic conditions, metabolism maintains a low intracellular concentration and, therefore, the net flux of adenosine is into cardiomyocytes and endothelial cells. Under such conditions, administration of transport inhibitors increases extracellular concentrations of adenosine, leading to vasodilatation. 8 However, increased adenine nucleotide breakdown and inhibition of adenosine kinase duri...

Ablation of nonmuscle myosin (NM) II-B and II-C in mice results in a defect in cardiac myocyte karyokinesis. More than 90% of the double knockout cardiac myocytes demonstrate defects in chromatid segregation and mitotic spindle formation. The requirement for NM II in karyokinesis is demonstrated in both the intact heart and in HL-1 atrial myocytes in culture.

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In patients with congestive heart failure, high serum levels of the proinflammatory cytokine interleukin (IL)-18 were reported. A positive correlation was described between serum IL-18 levels and the disease severity. IL-18 has also been shown to induce atrial natriuretic factor (ANF) gene expression in adult cardiomyocytes. Because re-expression of the fetal gene ANF is mostly associated with hypertrophy, a hallmark of heart failure, we hypothesized that IL-18 induces cardiomyocyte hypertrophy. Treatment of the cardiomyocyte cell line HL-1 with IL-18 induced hypertrophy as characterized by increases in protein synthesis, phosphorylated p70 S6 kinase, and ribosomal S6 protein levels as well as cell surface area. Furthermore, IL-18 induced ANF gene transcription in a time-dependent manner as evidenced by increased ANF secretion and ANF promoter-driven reporter gene activity. Investigation into possible signal transduction pathways mediating IL-18 effects revealed that IL-18 activates phosphoinositide 3-kinase (PI3K), an effect that was blocked by wortmannin and LY-294002. IL-18 induced Akt phosphorylation and stimulated its activity, effects that were abolished by Akt inhibitor or knockdown. IL-18 stimulated GATA4 DNA binding activity and increased transcription of a reporter gene driven by multimerized GATA4-binding DNA elements. Pharmacological inhibition or knockdown studies revealed that IL-18 induced cardiomyocyte hypertrophy and ANF gene transcription via PI3K, PDK1, Akt, and GATA4. Most importantly, IL-18 induced ANF gene transcription and hypertrophy of neonatal rat ventricular myocytes via PI3K-, Akt-, and GATA4-dependent signaling. Together these data provide the first evidence that IL-18 induces cardiomyocyte hypertrophy via PI3K-dependent signaling, defines a mechanism of IL-18-mediated ANF gene transcription, and further supports a role for IL-18 in inflammatory heart diseases including heart failure.

Rab1GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ERto-Golgi transport as a regulatory site for control of cardiomyocyte growth.

Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at -1095 and -770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O 2 ) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at -1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter.