A soluble neuronal factor alters contractile function of ventricular myocytes without effect on troponin T isoform expression.

OBJECTIVE: The purpose of this investigation was to establish a model system to facilitate identification of the sympathetic neuronal factor(s) that promotes improved contractility in neonatal cardiac myocytes. Conditioned medium from PC12 cells with sympathetic phenotype served as the source of the neuronal factor. METHODS: Contraction frequency, amplitude and velocity of cultured neonatal rat cardiac myocytes were measured by online video analysis. Interventions included in vitro sympathetic innervation, exposure to PC12 conditioned medium, neurotransmitters and antagonists. Metabolic activity was assayed by 2-deoxyglucose uptake. Troponin T isoform expression was analyzed by SDS-polyacrylamide gel electrophoresis. RESULTS: Medium conditioned by neuronal PC12 cells induced contractility changes similar to those induced by in vitro sympathetic innervation. These effects of PC12 conditioned medium and innervation were not suppressed by adrenergic or muscarinic antagonists nor reproduced by neuropeptide Y or somatostatin. Neuronal PC12 conditioned medium but not chromaffin PC12 conditioned medium, increased metabolic activity of the myocytes as detected by [3H]-2-deoxyglucose, indicating that the effect was specific to the neuronal PC12 cells. The in vitro switch of troponin T isoform expression was not altered by exposure to PC12 conditioned medium. CONCLUSIONS: Increased contractile function induced by sympathetic innervation is reproduced by PC12 conditioned medium, but neither is mediated by sympathetic or muscarinic neurotransmitters. Troponin T isoform expression is not related to the contractility changes. This model system will allow identification of the factor(s).

Differential growth of neonatal WKY and SHR ventricular myocytes within sympathetic co-cultures.

Sympathetic innervation is known to increase heart size in the immature animal, yet the mechanism for this growth remains to be established. This comparative study stereologically quantified the volume of cultured neonatal ventricular myocytes with and without in vitro sympathetic innervation to isolate the mechanisms regulating cardiac growth. Since ventricular myocyte size at birth differs between the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY), we questioned whether SHR myocytes respond differently than WKY myocytes to innervation. Four groups of ventricular myocytes from each strain were compared: myocytes grown alone, myocytes innervated by cultured sympathetic neurons, innervated myocytes exposed to adrenoceptor blockade, and non-innervated myocytes in co-culture dishes. Volumes for the myocyte, nucleus, cytoplasm, mitochondria, sarcomeres and other cellular organelles were assessed within each population and between populations. Relative volumes were determined for the mitochondria, sarcomeres, and other cellular components within the cytoplasm. Innervated WKY myocytes were 38% larger than control myocytes (P < 0.0004). This growth was not blocked by adrenoceptor blockade (P = 0.89 vs. innervated) and was present in the non-innervated myocytes distant from the neurons in the co-cultures (P = 0.39 vs. innervated). SHR myocytes were 36% larger than WKY myocytes (P < 0.009) but did not increase with innervation (P = 0.48). SHR myocyte size was also unaffected by adrenoceptor blockade (P = 0.39) or presence of the neurons in the culture dish (P = 0.53). Neonatal WKY ventricular myocyte growth can be provoked in vitro by sympathetic innervation via regulatory mechanisms independent of neuroeffector transmission or anatomic contact, whereas volume of neonatal SHR myocytes is unaltered by sympathetic coculture. These findings are significant for understanding normal as well as aberrant cardiomyocyte growth.

Application of stereological analysis of cell volume to isolated myocytes in culture with and without adrenergic innervation.

A three-dimensional analysis to evaluate structural changes in cultured cardiac myocytes following adrenergic innervation was performed using stereological techniques formerly limited to cells in tissue and organs. Cell volumes were calculated for two groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons. Relative and absolute volumes of the nucleus, cytoplasm, and cell were quantified by systematically sampling sections throughout the cell and by point count sampling techniques. Volumetric estimates were similarly determined for the mitochondria, sarcomeres, and other cellular components in the cytoplasm. Data were analyzed with ANOVA and randomized block design to control for variation among the cultures. Adrenergic innervation produced a 44% increase in cell volume, X +/- SEM, (3,344 +/- 196 microns3 to 4,816 +/- 400 microns3, P = 0.007). The absolute volume of mitochondria significantly increased after innervation (521 +/- 42 microns3 to 744 +/- 54 microns3, P less than 0.01). Absolute sarcomere volume did not change significantly (750 +/- 92 microns3 to 642 +/- 1061 microns3, P = 0.14). Other cellular components, defined as all cytoplasmic components except mitochondria and sarcomeres, significantly increased with innervation (1,739 +/- 166 microns3 to 3,097 +/- 338 microns3, P = 0.02). The relative volume of the nucleus and the cytoplasm in the cell remained unchanged following innervation. However, the relative volume of mitochondria decreased by 6%, the percent of the cytoplasm occupied by the sarcomeres decreased by 44%, and the volume occupied by the other cellular components increased by 22%. These findings support the use of stereological analysis as a means to quantify cell volumes of cultured myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of rat cardiac myocyte growth by a neuronal factor secreted by PC12 cells.

Sympathetic innervation of cardiac myocytes in vitro induces growth independent of anatomic contact between the neurons and myocytes and is not mediated by alpha- or beta-adrenergic receptor stimulation. To establish a model system that will allow purification and identification of the neuronal factor(s) responsible for mediating this regulation, we have initiated studies utilizing conditioned medium from the PC12 cell line. PC12 cells acquire a cholinergic sympathetic neuronal phenotype when exposed to nerve growth factor. Culture medium conditioned by neuronal PC12 cells, but not nonneuronal PC12 cells, induces growth in newborn rat cardiac myocytes as measured by surface area and [35S]methionine incorporation into protein and increases expression of atrionatriuretic peptide, a marker for myocyte hypertrophy. The magnitude of the growth response is dose-dependent and mimics the response to sympathetic innervation. The myocyte response to conditioned medium is not detectable after 24 h of exposure; maximal rate of protein synthesis is obtained within 48 h. Neuronally differentiated PC12 cell-conditioned medium stimulation of growth could not be mimicked by alpha- or beta-adrenergic agonists or muscarinic agonists, nor inhibited by alpha- or beta-adrenergic antagonists, nor by muscarinic antagonists. Neuropeptide Y and somatostatin, peptides known to be present in PC12 cells and sympathetic neurons, were also ineffective at reproducing the effect of neuronally differentiated PC12 cell-conditioned medium. These data indicate that neuronal cells release a soluble factor, different from neurotransmitter, which stimulates myocyte growth. They further identify the PC12 cell line as providing a convenient and abundant supply of this molecule, thus facilitating its further characterization.