Development of heart cells in culture: Studies using an affinity purified antibody to a myosin light chain

Klein, Irwin; Daood, Monica J.; Whiteside, Theresa L.

doi:10.1002/jcp.1041240109

Cited by 15 publications

(4 citation statements)

References 20 publications

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“…3 Approximately 85% of the cells were myocytes as determined by immunofluorescent staining with antimyosin antibodies, and the number of myocytes per culture dish did not change during KCl arrest. 28 Myocytes were maintained in a serumfree medium that contained 0.1 mM BrdU throughout the entire course of each experiment to prevent proliferation of nonmyocytic cells.…”

Section: Culture Of Kcl-arrested Myocytesmentioning

confidence: 99%

Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture.

McDermott

Morgan

1989

Circulation Research

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Neonatal ventricular myocytes that were incubated in a well-defined serum-free medium containing 50 mM KCl did not contract and maintained stable cell size, as assessed by the protein/DNA ratio. The present study utilized KCl-arrested cells to examine the effect of constant rates of synchronous contraction in normal [K+]o (4 mM) as a physiological stimulus for myocyte growth. Cell growth increased following the onset of contraction when measured over 3 days. The rate of protein synthesis was accelerated in parallel by contraction, but the rate of protein degradation remained similar to rates in noncontracting cells. The capacity for protein synthesis was estimated by total RNA content and was increased in contracting as compared with KCl-arrested cells. This increase was accompanied by faster rates of RNA synthesis as determined from the incorporation of [3H]uridine into RNA and the specific activity of the cellular UTP pool. The rate of RNA degradation was accelerated during contraction but the difference between the rates of RNA synthesis and degradation resulted in net RNA accumulation of 49% after 3 days. These data demonstrated that 1) contractile activity stimulated myocyte growth through an increased capacity for protein synthesis and 2) the increased capacity for protein synthesis involved acceleration of the rate of RNA synthesis. Since enhancement of protein synthetic capacity is a common feature of myocyte hypertrophy in vivo and in vitro, this model can be used to examine the regulation of ribosome synthesis during hypertrophic growth.

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Section: Culture Of Kcl-arrested Myocytesmentioning

confidence: 99%

Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture.

McDermott

Morgan

1989

Circulation Research

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“…From these observations, we determined that the adenoviruses and reagents were added at a time during which twitch contractions are able to influence cardiomyocyte growth and/or remodeling in vitro. 8,9 Once spontaneous contractions began, the average beat frequency was 1.0 AE 0.1 s À1 at room temperature and was not significantly different between the four groups.…”

Section: Resultsmentioning

confidence: 88%

“…1,2,4,6,7 In contrast, neonatal cardiomyocytes that are unable to contract have suppressed hypertrophic growth and reduced myofibril development, suggesting that a mechanotransduction mechanism may underlie this response. 8,9 Moreover, the stiffness of the cardiac extracellular environment, which provides a mechanical resistance to twitch contractions, can stimulate cardiomyocytes to improve their myofibril structure and amplify calcium transients in order to produce a more powerful twitch contraction. [10][11][12][13] Examples such as these suggest that a feedback mechanism exists in cardiomyocytes, enabling adaptation to their mechanical environment through remodeling of their myofibril structure.…”

Section: Introductionmentioning

confidence: 99%

Enhanced contractility with 2-deoxy-ATP and EMD 57033 is correlated with reduced myofibril structure and twitch power in neonatal cardiomyocytes

Rodriguez

Han

et al. 2013

Integr. Biol.

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As cardiomyocytes mature, their sarcomeres and Z-band widths increase in length in order for their myofibrils to produce stronger twitch forces during a contraction. In this study, we tested the hypothesis that tensional homeostasis is affected by altering myofibril forces. To assess this hypothesis, neonatal rat cardiomyocytes were cultured on arrays of microposts to measure cellular contractility. An optical line scanning technique was used to measure the deflections in the microposts with high temporal resolution, enabling the analysis of twitch force, twitch velocity, and twitch power. Myofibril force production was elevated by vector-mediated overexpression of ribonucleotide reductase (RR) to increase cellular dATP content or by adding the inotropic agent EMD 57033 (EMD). We found that RR and EMD treatment did not affect cardiomyocyte twitch force, but it did lead to reduced twitch velocity and twitch power. Immunofluorescent analysis of α-actinin revealed that RR-over-expressing cardiomyocytes and EMD-treated cardiomyocytes had lower spread area, sarcomere length, and Z-band width as compared to control cells. These results indicate a correlation between myofibril structure and cardiac power. This correlation was confirmed by exposing the cells to the myosin II inhibitor blebbistatin, and then subsequently washing it out. After wash-out, cardiomyocytes exhibited a reduction in twitch force, velocity, and power due to shorter sarcomere length and Z-band widths. Our results suggest that cardiac myofibril structure is regulated by tensional homeostasis. If myofibril-generated forces in cardiomyocytes are elevated, a state of tensional homeostasis is maintained by producing sufficient twitch forces with a lower degree myofibril structure.

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“…In cultured heart cells the role of contraction in regulating myosin subcellular organization has been studied (22). Addition of culture medium containing 50 mM KCI resulted in a complete arrest of otherwise actively twitching heart cells in culture.…”

Section: Discussionmentioning

confidence: 99%

Early cross-striation formation in twitching Xenopus myocytes in culture.

Kidokoro

Saito

1988

Proc. Natl. Acad. Sci. U.S.A.

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Spontaneous release of neurotransmitter has been demonstrated in various types of synapses. Its physiological significance, however, is still unknown. In nerve-muscle cultures of embryonic Xenopus laevis, we observed that acetylcholine, which is released spontaneously at the synaptic terminal, caused frequent twitches of muscle cells. These muscle cells developed cross-striations earlier than neighboring nontwitching cells. This effect of innervation was unaffected by tetrodotoxin but was blocked by a-bungarotoxin. Repeated iontophoretic application of acetylcholine or KCI to muscle cells caused twitches and also accelerated the formation of crossstriations. Thus twitching apparently promotes lateral alignment of myofibrils. It is also known that myosin synthesis is higher in twitching muscle cells. Therefore, successfully innervated twitching muscle cells may have an advantage for faster differentiation over neighboring non-twitching muscle cells. We suggest that spontaneously released transmitter may serve as a mediator for trophic interaction at forming synapses.Since the discovery of miniature endplate potentials at the neuromuscular junction (1), similar spontaneous release of synaptic transmitter has been observed in many other chemical synapses (2). Although the amplitude of miniature endplate potentials is small in adult muscle cells, at newly formed neuromuscular junctions it is considerably larger due to the higher input impedance of young myotubes (3). The amplitude of synaptic potentials increases as acetylcholine (AcCho) receptors accumulate at the postsynaptic membrane (4). Spontaneous synaptic potentials are detected soon after a growth cone contacts a muscle cell (5). In fact, the growth cone releases AcCho even before contacting a muscle cell (6, 7). The physiological significance of spontaneous release of transmitter is not yet known. AcCho thus released from growing neurites may serve as a mediator for trophic interaction. MATERIALS AND METHODSThe dorsomedial portion of Xenopus embryos at stage 14 (8) was dissected, and the covering skin was removed by using a pair of needles. Neurons and myocytes were dissociated in Ca2+-and Mg2+-free saline as described (9) and plated on the collagen-coated cover glass in culture medium [0.6x Dulbecco's modified Eagle's medium (buffered by Hepes to pH 7.4, GIBCO) supplemented with 5% (vol/vol) horse serum]. These cultures were maintained at room temperature (21-23°C) in the glass chamber.Xenopus myocytes are mononucleated and constitute tail musculature of the tadpole. Cultured myocytes are flat, and cross-striation is clearly seen under a phase-contrast microscope (40 x water-immersion objective, Zeiss). About 17 hr after plating, cultures were rinsed three times with culture medium to remove unattached cells. The great majority of attached cells were myocytes, and some ofthem possessed an adherent neuron. Typically a few thousand myocytes per dish attached to the substrate, and 20-40 neurons adhered to the myocytes.AcCho and K+ were applied iontoph...

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Development of heart cells in culture: Studies using an affinity purified antibody to a myosin light chain

Cited by 15 publications

References 20 publications

Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture.

Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture.

Enhanced contractility with 2-deoxy-ATP and EMD 57033 is correlated with reduced myofibril structure and twitch power in neonatal cardiomyocytes

Early cross-striation formation in twitching Xenopus myocytes in culture.

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