Single Rat Heart Research Articles

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart

Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.

High Resolution Top-Down MS/MS Reveals Single Amino Acid Sequence Polymorphisms in Rat Cardiac Troponin

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex. Two of the three subunits, cTnI and cTnT, are expressed only in cardiac muscle and are widely used in the clinic as serum biomarkers of cardiac injury. cTnI and cTnT are subject to extensive post-translational modification such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. In order to obtain a global view of the state of post-translational modification of cTn, we are performing high resolution top-down mass spectrometry on cTn subunits isolated from native tissues. Whole cTn complexes affinity purified from a single rat heart were analyzed in a 7 Tesla Thermo LTQ-FT-ICR mass spectrometer equipped with an ESI source. High resolution MS spectra of cTn from healthy adult rats showed molecular ions for intact cTnT and cTnI as well as phosphorylation and acetylation patterns similar to human cTnI (Zabrouskov et al., 2008 Mol Cell Proteomics, in press). ‘Shadow peaks’ of similar intensity to parent peaks were detected exhibiting masses of cTnI + 16 Da and cTnT + 128 Da, suggestive of single amino acid polymorphisms. Tandem mass spectrometry (MS/MS) analysis by ECD and CAD fragmentation of intact and protease-digested cTn subunits localized an Ala/Ser polymorphism at residue 7 of cTnI, and an additional Gln within a 3 residue alternative splice site beginning at residue 192 of cTnT. High resolution top-down MS/MS has revealed intriguing heterogeneity not only in the extent of phosphorylation, but also in amino acid sequences of cTnI and cTnT even within a single rat heart. Supported by NIH, AHA, UW-CVRC & Wisconsin Partnership for a Healthy Future.

Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry.

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 +/- 3%, 46 +/- 1%, 13 +/- 3%, respectively. cTnT was predominantly monophosphorylated and partially proteolyzed at the Glu(29)-Pro(30) peptide bond. Also observed in high resolution spectra were 'shadow' peaks of similar intensity to 'parent' peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.

Plasma‐membrane K ATP channel‐mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia

Our recent data demonstrate that activation of pmKATP channels polarizes the membrane of cardiomyocytes and reduces Na+/Ca2+ exchange-mediated Ca2+ overload. However, it is important that these findings be extended into contractile models of hypoxia/reoxygenation injury to further test the notion that pmKATP channel activation affords protection against contractile dysfunction and calcium overload. Single rat heart right ventricular myocytes were enzymatically isolated, and cell contractility and Ca2+ transients in field-stimulated myocytes were measured in a cellular model of metabolic inhibition and reoxygenation. Activation of pmKATP with P-1075 (5 microM) or inhibition of the Na+/Ca2+ exchanger with KB-R7943 (5 microM)reduced reoxygenation-induced diastolic Ca2+ overload and improved the rate and magnitude of posthypoxic contractile recovery during the first few minutes of reoxygenation. Moreover,diastolic Ca2+ overload and posthypoxic contractile dysfunction were aggravated in ventricular myocytes either subjected to specific blockade of pmKATP with HMR1098 (20 microM) or expressing the dominant-negative pmKATP construct Kir6.2(AAA) in the presence of P-1075. Our results suggest that a common mechanism, involving resting membrane potential-modulated increases in diastolic [Ca2+]i, is responsible for the development of contractile dysfunction during reoxygenation following metabolic inhibition. This novel and highly plausible cellular mechanism for pmKATP-mediated cardioprotection may have direct clinical relevance as evidenced by the following findings: a hypokalemic polarizing cardioplegia solution supplemented with the pmKATP opener P-1075 improved Ca2+ homeostasis and recovery of function compared with hyperkalemic depolarizing St. Thomas' cardioplegia following contractile arrest in single ventricular myocytes and working rat hearts. We therefore propose that activation of pmKATP channels improves posthypoxic cardiac function via reductions in abnormal diastolic Ca2+ homeostasis mediated by reverse-mode Na+/Ca2+ exchange.

Substrate and inhibitor specificities of the monocarboxylate transporters of single rat heart cells.

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat heart cells these inhibitors are not effective at blocking lactate transport totally unless used at very high concentrations. Both carriers were inhibited by 3-isobutyl-1-methylxanthine (Ki 340 microM) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT-1). The overall Michaelis constant (Km) and maximum reaction rate (Vmax) for transport of a variety of substituted monocarboxylates (C2-C5) were determined, although it was not possible to elucidate the kinetic parameters of the two isoforms. Of physiological interest, the ketone bodies D-beta-hydroxybutyrate and acetoacetate had K(m) values of 10 and 5.4 mM, respectively. Vmax values were similar to those of L-lactate and pyruvate and indicate that transport could limit rates of utilization of ketone bodies. No stereoselectivity for L-over D-isomers of 2-chloro or 2-hydroxy acids was observed.

α1‐Adrenoceptors in the conduction system of rat hearts

1. We have characterized alpha 1-adrenoceptor in the conduction systems of the rat heart by quantitative autoradiography. 2. Consecutive 20 micron thick sections from a single rat heart containing the sinoatrial (SA) node and atrioventricular (AV) node were incubated with increasing concentrations of [3H]-prazosin with or without 10 microM phentolamine. After exposure to 3H-Ultrofilm, optical densities corresponding to the SA node and AV node were determined by computerized densitometry after comparison with 3H standards. 3. The SA node and AV node were stained heavily for cholinesterase and they contained a higher concentration of alpha 1-adrenoceptors than the adjacent myocardium without a significant change in the affinity. 4. These results support the hypothesis that alpha 1-adrenoceptors may play an important role not only in inotropism but also in chronotropism of rat hearts.

Inhibitory effects of cocaine on Ca2+ transients and contraction in single cardiomyocytes

The effects of cocaine on the Ca2+ transient responsible for excitation-contraction coupling were studied in single rat heart cells loaded with the fluorescent Ca2+ indicator fura 2. A high-speed imaging technique using a charge-coupled device as detector and transient image store [O'Rourke et al., Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H230-H242, 1990] was used to measure cytosolic free Ca2+ concentration ([Ca2+]i) and contraction simultaneously from the images of fluorescence. Cardiomyocytes maintained a basal [Ca2+]i of approximately 140 nM in presence or absence of cocaine. After electrical field stimulation, [Ca2+]i increased to a peak of 498 +/- 25 nM under control conditions. This was reduced to a peak [Ca2+]i of 389 +/- 25 nM after treatment with 50 microM cocaine. Cocaine also reduced the rate of rise of [Ca2+]i but did not affect the time to peak or the half time for resequestration of the released Ca2+. The rate and extent of cell shortening was reduced by cocaine in parallel with the inhibition of the [Ca2+]i transient. Cocaine had no effect on the half time for relaxation. Cocaine did not modify the relationship between contraction and the elevation of [Ca2+]i over a range of extracellular Ca2+ concentrations. The intracellular pool of Ca2+ releasable by caffeine was also unaffected by cocaine. In the presence of the beta-adrenergic agonist isoproterenol, which caused a large enhancement of peak [Ca2+]i and contraction, cocaine still inhibited both parameters. However, cocaine did not reverse the ability of isoproterenol to enhance the rate of Ca2+ reuptake and cell relaxation. Whole cell voltage-clamp studies showed that 50 microM cocaine reduced both the Na+ current (50%) and the Ca2+ current (30%). These data suggest that sarcolemmal ion channels are the primary site that, in cardiac muscle, mediate the negative inotropic effects and the suppression of [Ca2+]i transients by cocaine.

Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes

We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca 2+-transport and Ca 2+ K + -ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca 2+ K + -ATPase activity of 371 ± 55 nmol/min/mg protein (mean ± s.e.m.; n = 5) and an oxalate-stimulated Ca 2+-uptake activity of 103 ± 4 nmol/min/mg protein (mean ± s.e.m.; n = 6). Pretreatment of the SR vesicles with 5 μM ruthenium red increased the oxalate-stimulated Ca 2+-uptake to 204 ± 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca 2+-release channel), 100 ( Ca 2+ K + -ATPase ), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca 2+-binding protein bands in this preparation at 53–55 kDa (calsequestrin and/or calreticulin) and 97–100 kDa ( Ca 2+ K + -ATPase ). The presence of phospholamban, a regulatory protein of the Ca 2+ K + -ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [ 32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.

Effect of Halothane on Arrhythmogenic Responses Induced by Sympathomimetic Agents in Single Rat Heart Cells

The combination of catecholamines and halothane has long been recognized as arrhythmogenic. The purpose of this study was to evaluate whether the mechanism of this interaction originates at the single cell level. The incidence of spontaneous contractile waves occurring between stimulated beats (interbeat waves), early aftercontractions, and late aftercontractions was measured in rat myocytes exposed to sympathomimetics with and without halothane. Each of these endpoints in single cells has the potential to produce arrhythmias in multicellular preparations. Interbeat waves and late aftercontractions were observed with isoproterenol (1 X 10(-7) M) and norepinephrine (1-3 X 10(-7) M). The incidence of these phenomena was significantly reduced in the presence of 0.30 mM halothane. Early aftercontractions occurred in the presence of isoproterenol (1 X 10(-7) M), norepinephrine (1-3 X 10(-7) M), and phenylephrine (5-10 X 10(-6) M). There was a statistically significant decrease in the incidence of early aftercontractions in the presence of 0.30 mM halothane. These results indicate that the mechanism behind the clinically observed increased arrhythmogenicity of catecholamines with halothane does not arise at the level of single ventricular cells because halothane inhibited sympathomimetic-induced arrhythmogenic activity in this model. The probable mechanisms rather include altered impulse propagation, which might lead to phenomena such as reentry.

Extracellular ATP mediates an increase in H+ and Ca2+ ions associated with automaticity in a single rat heart cell

Extracellular ATP mediates an increase in H+ and Ca2+ ions associated with automaticity in a single rat heart cell

Changes in the calcium current of rat heart ventricular myocytes during development.

1. Calcium current (ICa) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult). 2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr. 3. In adult single cells, the application of ryanodine (10 microM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa. Ryanodine also produced a 13 mV shift in f infinity towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d infinity. In neonatal single cells, ryanodine (10 microM) had no significant effect on contraction, ICa, d infinity, f infinity, or gr. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift of f infinity to more positive potentials was seen. A large additional shift of f infinity was observed when ryanodine (10 microM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on f infinity and the application of ryanodine had no effect. 5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974; Hirakow & Gotoh, 1975; Ishikawa & Yamada, 1975; Legato, 1975; Hoerter, Mazet & Vassort, 1981). 6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.

A patch clamp study of the action of ethacyzine on the rapid inward sodium current in single rat heart muscle cells

The effect of ethacyzine (a diethylamine analogue of ethmozine) on the rapid inward sodium current (I Na) was studied in single rat ventricular muscle cells by a patch clamp technique. Extracellular application of 3 μM ethacyzine depressed the I Na in a use-dependent fashion. Rest recovery from the use-dependent block by ethacyzine followed an exponential time course with a time constant of about 215 ± 40 s (n = 4, mean ± S.E.).

A simplified method for the rapid isolation of cardiac intercalated discs

A simple and rapid method for the isolation of intercalated discs from a single rat heart is described. The outstanding features of this method are (i) a large quantity of starting material is not necessary, (ii) ultracentrifugation and separating gradient steps are not required, (iii) the disc fraction is relatively pure as verified by electron microscopy, and (iv) the starting tissue is rapidly homogenized into buffer, reducing possible damage to membrane structures after animal death. The fraction is rich in intercellular junctions, with gap junctions, fasciae adherentes and maculae adherentes all appearing well preserved. It should prove a useful starting base for further purification of these junctions and of sarcolemma from a specific portion of the cell surface membrane, namely the major area of structural interaction between adjacent cardiac cells.

Patch-voltage-clamp method for measuring fast inward current in single rat heart muscle cells.

A patch-voltage-clamp method was used to measure fast inward ionic currents in single heart muscle cells. 1. Theoretical analysis including computer simulation has shown that the method provides fast settling of membrane potential (within 10 microseconds) and reliable voltage clamp on the tested membrane patch when the area of the patch is 200-300 times smaller than the area of the whole cell membrane. 2. The transient time at the I-V converter output is increased up to 70-75 microseconds due to the stray capacity in the I-V converter feedback. When fast-response operational amplifiers are used in the set up this transient time may be decreased to 30 microseconds by partial restoration of the high-frequency components of the signal. 3. Experimental data have shown that the ionic channel population in the membrane patch of about 5 micrometers in diameter is on the one hand large enough to directly observe integral ionic current and on the other hand small enough for the fluctuations of ionic current to be appreciable. This permits the method to be applied to ionic current investigations both by classical methods and by statistical analysis.

Sodium current in single rat heart muscle cells.

1. Rapid inward Na current (INa) was studied in isolated cells from rat ventricular myocardium by a double-suction-pipette voltage clamp technique. All experiments were carried out at 20-22 degrees C. 2. INa elicited by single depolarizing voltage steps from a holding potential, VH, of -80 mV had a threshold between -70 and -60 mV and was maximal at -30 to -20 mV. Peak currents in Krebs-Ringer solution containing 145 mM Na were of the order 0.9-1.8 mA cm-2, assuming an average cell surface area of 8000 square micrometers. 3. The reversal potential for INa was predicted by the Nernst equation for external Na in the range 1.45-145 mM with 16 mM-Na solution perfusing the interior of the cell. 4. Instantaneous I-V plots were linear for potentials of -100 to + 10 mV. Maximum Na conductance (-gNa) was calculated to be 25 mS cm-2 in 145 mM-Na solutions and gNa was constant for potentials positive to -10 mV. 5. INa activated with a time constant of 0.7 msec at -55 mV, decreasing to 100 microsec on depolarizations positive to + 10 mV. 6. Two time constants (tau h1, tau h2) were required to describe INa inactivation during a maintained depolarization, with tau h2 three to four times as long as tau h1. tau h1 was about 2 msec at -50 mV, decreasing to 0.9 msec at -10 mV. 7. The time course for recovery of INa from inactivation also exhibited two time constants (tau r1, tau r2), with the longer tau r2 having a maximum value of the order 100 msec in the potential range -60 to -80 mV. 8. INa in isolated rat cardiac cells has a low sensitivity to tetrodotoxin, requiring a concentration of 30 micrometers for complete blockade.

The rates of growth of protein fractions of the heart and kidney of the rat

The procedure for fractionating the proteins of a single rat heart has been modified for the kidney. The quantity of each protein fraction of the heart and kidney has been studied for female rats of the Addis-Slonaker strain, ranging in age from 4 weeks to 1 year. Regressions were derived correlating the quantity of protein of each fraction with the weight of the rat. The growth rate of each fraction was calculated. It was observed that there were wide individual differences. For example α-myosin and β-myosin in the heart grow at a greater rate than other protein fractions. It is suggested that the rate of growth of a particular protein fraction may be dependent upon the demands placed upon it.