Cardiac myocytes are activated by hormonal and mechanical signals and respond in a variety of ways, from altering contractile function to inducing cardio-protection and growth responses. The use of genetic mouse models allows one to examine the role of cardiac-specific and other genes in cardiac function, hypertrophy, cardio-protection, and diseases such as ischemia and heart failure. However, studies at the cellular level have been hampered by a lack of suitable techniques for isolating and culturing calcium-tolerant, adult mouse cardiac myocytes. We have developed a straightforward, reproducible protocol for isolating and culturing large numbers of adult mouse cardiac myocytes. This protocol is based on the traditional approach of retrograde perfusion of collagenase through the coronary arteries to digest the extracellular matrix of the heart and release rod-shaped myocytes. However, we have made modifications that are essential for isolating calcium-tolerant, rod-shaped adult mouse cardiac myocytes and maintaining them in culture. This protocol yields freshly isolated adult mouse myocytes that are suitable for biochemical assays and for measuring contractile function and calcium transients, and cultured myocytes that are suitable for most biochemical and signaling assays, as well as gene transduction using adenovirus.

SUMMARY Proliferating nonmyocardial cells (NMCs) complicate primary heart cultures and may influence myocardial cell (MC) differentiation. In cultures from the day-old rat ventricle, we validated a method to arrest this proliferation; and we quantitated cross-striated cells and the chronotropic response to isoproterenol to assess MC differentiation. MCs were cultured at single cell density using an improved method. By 4 days, all cells could be identified as MCs or NMCs. In cultures treated for 3 days with bromodeoxyuridine (BrdU), 0.1 miw, serial cell counts were unchanged through 11 days. Among 50,000 cells from 110 cultures, 75-80% were MCs. In control cultures without BrdU, NMC density was 3-and 6-fold that in BrdU-treated cultures at days 5 and 8, respectively (P < 0.01), although the MCs were not overgrown. The MCs did not proliferate in either culture. The proportion of living MCs with cross-striations was similar in treated and control cultures at day 5 (72.6% vs. 69.9%, P > 0.05) but was lower in controls at day 8 (86.3% vs. 50.4%, P < 0.01). A sensitive (ED 50 10-100 pM), specific chronotropic response to L-isoproterenol was present in both treated and control cultures, but the maximum response was only 20-30% as great in controls on days 4 and 8 (P < 0.01). Baseline beating rates were the same. The MCs had well-differentiated ultrastructure, including a T system. By autoradiography, a maximum 18.4% of MCs had nuclear incorporation of 3 H-BrdU at day 8. Media conditioned by NMCs or by the control cultures did not change the cross-striations or isoproterenol response of BrdU-treated cultures. Thus, in a new culture preparation with few and stable NMCs, morphological and functional MC characteristics were different from those of MCs in cultures with proliferating NMCs. We suggest that an MC-NMC interaction can alter MC properties and that this effect should be considered in studies of primary rat heart cultures. The pure, stable, well-differentiated MCs in BrdUtreated cultures will be useful for studying MC growth, repair, and other time-dependent phenomena.

ulated hypertrophy of cultured rat myocardial cells is an alpha, adrenergic response.

Catecholamines may be one of the molecular signals linking increased circulatory demand to myocardial hypertrophy, and I have found previously that norepinephrine stimulates hypertrophy of cultured neonatal rat heart muscle cells through an alpha 1-adrenergic receptor. Since catecholamine stimulation of contractility is believed to be under beta-adrenergic control, I asked whether these cultured heart cells had dual pathways regulating growth and contractility through alpha- and beta-adrenergic receptors, respectively. I examined the effect of adrenergic agents on hypertrophy and beating of myocytes in serum-free cultures. Hypertrophy was defined as an increase in myocyte surface area and in cell protein content, measured by a radioisotopic method, and chronotropic activity was examined visually. Norepinephrine and epinephrine were equipotent stimulants of hypertrophy and beating, increasing cell protein and area 1.5- to 2-fold, and the proportion of beating cells from 5% or less to 95%. Response maxima occurred 24-48 hours after exposure, and EC50 were 20-200 nM. Studies with other agonists (phenylephrine, methoxamine, clonidine, isoproterenol, dopamine) and antagonists (prazosin, terazosin, yohimbine, propranolol, betaxolol, ICI 118,551) indicated that hypertrophy was mediated through an alpha 1-adrenergic receptor, whereas the induction of beating required both alpha 1- and beta 1-receptor activation. Hypertrophied cells with minimal beating were produced by alpha-stimulation, alone. In contrast, alpha-plus beta-stimulation in the presence of cycloheximide to inhibit protein synthesis resulted in maximum beating but no hypertrophy. These findings imply that growth and beating can be regulated independently through separate cellular pathways.

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

Background-In ␣1-AR knockout (␣1ABKO) mice that lacked cardiac myocyte ␣1-adrenergic receptor (␣1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte ␣1-ARs and whether the ␣1A, ␣1B, or both subtypes mediated protection. Therefore, we investigated ␣1A and ␣1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of ␣1-ARs in cardiac myocytes. Methods and Results-We cultured ␣1ABKO myocytes and reconstituted ␣1-AR signaling with adenoviruses expressing ␣1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H 2 O 2 and was measured by annexin V/propidium iodide staining. In ␣1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the ␣1A subtype, but not the ␣1B, rescued ␣1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined ␣1-AR activation of extracellular signal-regulated kinase (ERK). In ␣1ABKO hearts, aortic constriction failed to activate ERK, and in ␣1ABKO myocytes, expression of a constitutively active MEK1 rescued ␣1ABKO myocytes from norepinephrine-induced death. In addition, only the ␣1A-AR activated ERK in ␣1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked ␣1A survival signaling in ␣1ABKO myocytes. Conclusions-Our results demonstrate a direct protective effect of the ␣1A subtype in cardiac myocytes and define an ␣1A-ERK signaling pathway that is required for myocyte survival. Absence of the ␣1A-ERK pathway can explain the failure to activate ERK after aortic constriction in ␣1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

SUMMARY. The role of hormones and other humoral factors in the regulation of myocardial hypertrophy has been difficult to evaluate. We asked whether myocardial cell hypertrophy could be demonstrated in cultures from the day-old rat ventricle and evaluated the effect of serum concentration and catecholamines on the growth process. Two single-cell preparations were used: serumsupplemented, bromodeoxyurdine-treated cultures and serum-free cultures with transferrin and insulin. Both preparations were characterized by myocardial cell predominance (about 75-80% of total cells) and constant cell numbers. Myocardial cell size was documented by photomicroscopy and quantified by volume (microscopic diameter of suspended cells), surface area (planimetry of attached cells), and total cell protein concentration (Lowry method and cell counts). Growth was also evaluated in pure nonmyocardial cell cultures. In cultures with 5% (vol/vol) serum, myocardial cell size increased 2-to 3-fold over 11 days in culture. Final volume, surface area, and protein concentration were about 3000 jum 3 /cell, 5000 /nmVcell, and 1500 pg/cell, respectively. Serum had a dose-related effect on myocardial cell hypertrophy; myocardial cell size increased about 4-fold when serum concentration was increased from 0% to 5% or 10%. Cells maintained in serum-free medium with transferrin and insulin (each 10 fig/ml) did not hypertrophy, but did remain responsive to the growth-promoting activity of serum. Chronic exposure to isoproterenol or norepinephrine (1 /IM) significantly stimulated myocardial cell hypertrophy. This stimulation was dose-related, was not blocked by equimolar propranolol, was not associated with a sustained chronotropic effect, and was more pronounced in the serum-free preparation. In pure cultures of nonproliferating (bromodeoxyuridine-treated) nonmyocardial cells, cell size also increased with time in culture, but variation in serum concentration and addition of norepinephrine had no significant effect on cell size. Myocardial cell hypertrophy occurs in culture and is regulated by variations in the culture medium, including serum, with its contained hormones and growth factors, and catecholamines. The culture preparation can be used to explore the regulation of myocardial cell hypertrophy by nonhemodynamic factors. (Ore Res 51: 787-801, 1982) VERY little is known about the role of hormones or other humoral factors in myocardial hypertrophy (Cohen, 1974). Experiments in vivo are complicated by hemodynamic, hormonal, and neural reflexes elicited by endocrine ablations or hormone treatments. In vitro models such as the isolated perfused heart or the heart in organ culture offer greater experimental control, but growth does not occur in these preparations (Morkin, 1974;Wildenthal et al., 1976). Cell cultures have been widely used to study the regulation of cell proliferation, and it has recently been proposed (Clark and Zak, 1981;Frelin, 1980;Speicher et al., 1981) that heart cells in primary culture may be a useful model system for stud...

Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of non-coding sequences highly enriched in human heart enhancers which is likely to facilitate down-stream studies of the role of enhancers in development and pathological conditions of the heart.