Rat Neonatal Myocytes Research Articles

Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

Free calcium transients in spontaneously beating rat neonatal myocytes measured by digitized video microscopy

Free calcium transients in spontaneously beating rat neonatal myocytes measured by digitized video microscopy

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes.

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly. In contrast, 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin II enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

Biochemical and subcellular distribution of arachidonic acid in rat myocardium.

Selective release of arachidonic acid from prelabeled phospholipid pools has been observed following exposure of neonatal rat cardiac myocytes to metabolic inhibitors in vitro and has been correlated temporally with the development of irreversible sarcolemmal damage. Hydrolysis of phospholipids with release of arachidonic acid may be an important mechanism in the pathogenesis of sarcolemmal damage induced by ischemia. To elucidate potential subcellular loci of arachidonic acid release in ischemic myocardium, we characterized the phospholipid composition of adult rat myocardial sarcolemma and delineated the biochemical and subcellular distribution of radiolabeled arachidonic acid in neonatal rat myocytes incubated with [3H]-arachidonic acid for selected intervals. Although arachidonic acid comprised 13 +/- 3% of total aliphatic moieties in combined choline and ethanolamine glycerophospholipids isolated from purified adult rat myocardial sarcolemma, radiolabeled arachidonic acid could not be detected in sarcolemma of cultured neonatal rat myocytes incubated with 5 nM [3H]arachidonic acid for 24 h. Radioactivity was located almost exclusively in mitochondria and internal cytoplasmic membranes (primarily sarcoplasmic reticulum), which collectively contained 90% of myocyte radioactivity. These results indicate that radiolabeled arachidonic acid released from prelabeled phospholipid pools on exposure of neonatal rat myocytes to oxidative inhibitors is derived from mitochondria and internal cell membranes. The diminutive labeling of the sarcolemma suggests that turnover of arachidonoyl phospholipids is slower in the sarcolemma than in other membranous organelles.

The dependence of electrophysiological derangements on accumulation of endogenous long-chain acyl carnitine in hypoxic neonatal rat myocytes.

To determine whether accumulation of long-chain acyl carnitine contributes to electrophysiological abnormalities induced by hypoxia, we characterized effects of normoxic and hypoxic perfusion on the subcellular distribution of endogenous long-chain acyl carnitine and transmembrane potentials of cultured rat neonatal myocytes. Hypoxia increased long-chain acyl carnitine more than 5-fold. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (10 microM), a carnitine acyltransferase inhibitor, precluded accumulation of long-chain acyl carnitine induced by hypoxia. Tissue was processed for electron microscopy by a procedure specifically developed for selective extraction of endogenous short-chain and free carnitine but retention of endogenous long-chain acyl carnitine. In normoxic-perfused cells, long-chain acyl carnitine was concentrated in mitochondria and cytoplasmic membranous components. Only small amounts were present in sarcolemma. Hypoxia increased mitochondrial long-chain acyl carnitine by 10-fold and sarcolemmal long-chain acyl carnitine by 70-fold. After 60 minutes of hypoxia, sarcolemma contained 1.4 X 10(7) long-chain acyl carnitine molecules/micron 3 of membrane volume, a value corresponding to approximately 3.5% of total sarcolemmal phospholipid. Hypoxia also significantly decreased maximum diastolic potential, action potential amplitude and maximum upstroke velocity of phase 0. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate inhibited accumulation of long-chain acyl carnitine in each subcellular compartment and prevented the depression of electrophysiological function induced by hypoxia. These results strongly implicate endogenous long-chain acyl carnitine as a mediator of electrophysiological alterations induced by hypoxia.

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor.

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.

Characterization of a potentially reversible increase in beta-adrenergic receptors in isolated, neonatal rat cardiac myocytes with impaired energy metabolism.

Previous studies have reported that the numbers of beta- and alpha-adrenergic receptors increase in ischemic myocardium. In vivo studies have raised questions regarding the mechanisms involved in the adrenergic receptor alterations and the consequences of these alterations. The purpose of this study was to evaluate potential relationships among beta-adrenergic receptor changes, high energy phosphate reduction, and severity of cell injury in cultured neonatal rat myocytes treated with metabolic inhibitors. The potential for reversal of the receptor changes also was addressed. Binding parameters were measured using [125I]iodocyanopindolol. After 4 hours incubation in potassium cyanide and 2-deoxyglucose, there was a 43% increase in beta-adrenergic receptor number, 41% decrease in adenosine triphosphate, and minimal morphological change in myocytes. Twenty-four hours after removal of the inhibitors, myocytes exhibited a return to normal of the receptor number and adenosine triphosphate level. Iodoacetate treatment for up to 3 hours resulted in marked reduction in adenosine triphosphate and increasing severity of cell injury. The number of beta-adrenergic receptors was unchanged at 1.2 hours, increased at 1.5-2 hours, and decreased at 3 hours. Thus: beta-adrenergic receptor density increases during relatively early stages of injury in metabolically impaired myocytes with reduced adenosine triphosphate levels and decreases subsequently, after the myocytes become irreversibly injured; the increased beta-adrenergic receptor density in moderately injured myocytes can be reversed upon removal of the injurious agent and restoration of the cellular adenosine triphosphate level; and changes in catecholamines mediated by an intact nervous system are not required for an increase in beta-adrenergic receptor density in the setting of impaired energy metabolism.

Neuronal regulation of the development of the alpha-adrenergic chronotropic response in the rat heart.

During development, there are changes in the response of automatic cardiac fibers to alpha-adrenergic agonists. In neonatal rat ventricle, in vitro phenylephrine (1 X 10(-8) M) induces an increase in automatic rate from 115 +/- 12 (mean +/- SEM) to 168 +/- 10 beats/min, P less than 0.05. In contrast, in adult rat ventricle, the rate decreases from 36 +/- 8 to 12 +/- 12 beats/min, P less than 0.05. At both ages, the response is attenuated by the alpha 1-antagonist, prazosin (1 X 10(-6) M). We used cultures of neonatal rat myocytes to determine whether maturation of innervation contributes to the ontogeny of this response. All non-innervated cultures showed a positive chronotropic response to alpha-stimulation; phenylephrine (1 X 10(-8) M) increased the rate from 40 +/- 2 to 52 +/- 2 beats/min, P less than 0.05. In contrast, 60% of the myocytes innervated with sympathetic neurons showed a decrease in rate in response to phenylephrine, from 78 +/- 6 to 67 +/- 6 beats/min, P less than 0.05. The negative chronotropic effect of phenylephrine did not result from the release of acetylcholine or adenosine, or the inhibition of presynaptic norepinephrine release by phenylephrine. Furthermore, exposure to neuronal norepinephrine is not responsible for the alteration in muscle cell responsiveness. In conclusion, we have demonstrated the modulation of the myocardial response to alpha-adrenergic stimulation by the occurrence of innervation in tissue culture. This provides an explanation for the previously identified ontogenetic change in alpha-adrenergic effects on intact cardiac fibers from excitation to inhibition.

Transport of 42K +, 201Tl + and [ 99mTc(dmpe) 2 · Cl 2] + by neonatal rat myocyte cultures

Transport of 42K +, 201Tl + and [ 99mTc(dmpe) 2 · Cl 2] + by neonatal rat myocyte cultures

A fixation procedure suitable for autoradiography of endogenous long-chain acyl carnitine.

A tissue processing procedure was evaluated for fixation of endogenous long-chain acyl carnitine (LCA) to facilitate autoradiographic subcellular localization of this amphiphile. Suspensions of neonatal rat myocytes labeled with exogenous 14C-palmitoyl carnitine retained 85.2% of the radiolabel after tissue processing. Autoradiography demonstrated no significant translocation of radiolabeled LCA from myocytes to unlabeled sheep erythrocytes mixed in equal proportions and processed together. To evaluate endogenous LCA fixation, cultured myocytes were incubated for 3 days with 3H-carnitine. Radioactivity was distributed in LCA, short-chain acyl carnitine, and free carnitine pools in proportion to the physiological concentrations of the metabolites traced. Before tissue processing, LCA contained 4.5% of total radioactivity. After tissue processing, labeled water-soluble components were lost and 88% of the retained radioactivity was in the LCA pool. The enrichment of endogenous LCA radioactivity was attributable to the selective extraction of endogenous short-chain and free carnitine. Nearly 75% of endogenous LCA was preserved. In contrast, 99.5% of both endogenous short-chain and free carnitine were extracted. Thus, endogenous LCA can be selectively preserved, permitting quantitative subcellular localization of this amphiphile with ultrastructural autoradiography.

Specific attachment of collagen to cardiac myocytes: In vivo and in vitro

The formation and attachment of collagen to the sarcolemma of cardiac myocytes were examined in vivo in neonatal rats and hamsters and in vitro in cultures of neonatal rat myocytes. Scanning, transmission, and high-voltage electron microscopy were used to show that the collagen struts attach to specific sites on the sarcolemma just lateral to the Z band of neonatal animals. In vitro, collagen preferentially attaches to distal end of myocytes at a site where internal stress fibers also attach to the sarcolemma. Formation of the collagen struts appeared to be a multistep process involving several components of the extracellular matrix. The role of the collagen struts is involved in the distribution of force generated by muscle contraction.

Ultrastructure of terminally differentiated adult rat cardiac muscle cells in culture.

Ventricular cardiac muscle cells isolated from adult rats were maintained in culture for 28 to 60 days and examined by transmission electron microscopy. In order to elucidate the detailed ultrastructure of the cultured myocytes, several different electron-dense stains were used. These included tannic acid, osmium ferrocyanide, osmium tetroxide (applied as a primary fixative), and lanthanum chloride, as well as more widely used stains such as osmium tetroxide, uranyl acetate, and lead citrate. Our results show that, compared to cultured neonatal rat myocytes, cultured myocytes derived from adult rats more closely resemble in vivo adult ventricular cells. The cultured adult myocytes contained typically distributed organelles such as nuclei, mitochondria, and elements of the sarcoplasmic reticulum. Myofilaments were well organized, and typical intercalated disks were observed between adjacent cells. Unlike cultured neonatal myocytes, the adult cells contained numerous residual bodies and a relatively well developed transverse tubular system. The transverse tubular system was identified by its continuity with the extracellular space (as indicated by the penetration of electron-dense extracellular tracers), location at or near the Z line, large lumenal diameter, and frequent participation in couplings with elements of the sarcoplasmic reticulum.

T-tubes in cultured mammalian myocardial cells.

T-tubes are among the last structural elements of the mammalian myocyte to develop in vivo. We were able to identify T-tubes in early cultures of neonatal rat myocytes. Ventricles were excised from 3- to 4-day-old neonatal rats, incubated overnight in cold trypsin, and treated with sequential changes of collagenase-hyaluronidase. Fractions of cells isolated in this manner were pooled and cultured in plastic petri dishes. In cells prepared for transmission electron microscopy, T-tubes were observed at the cell periphery of cultured myocytes, but were more difficult to identify as the cultures aged and became overgrown by fibroblasts. T-tubes were identified by virtue of their continuity with the sarcolemma, their relatively large diameter, and their regular entry at the level of the Z line. Even at optimal culture ages, T-tubes were not present in every myocyte. At the times T-tubes could be located, myocytes were beating and had begun to establish intercalated discs and gap junctions. The de novo formation of T-tubes in cultured myocytes of neonatal rat heart reflects a duplication of in vivo differentiation by the cultured myocyte. The appropriateness of cultured myocytes in the study of the development and physiology of the heart is emphasized by the in vitro formation of T-tubes.