Deterioration of beta-adrenergic modulation of cardiovascular function with aging.
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Aging↗
Biomedical subjects
E G Lakatta
Publications and source records attributed to E G Lakatta.
Spontaneous sarcoplasmic reticulum Ca2+ release leads to heterogeneity of contractile and electrical properties of the heart.
The cytosolic Ca2+ (Cai) oscillation generated by the sarcoplasmic reticulum (SR) in response to an action potential (AP) occurs relatively synchronously within and among cells. The SR can also generate spontaneous Cai oscillations (S-CaOs), i.e., not triggered by sarcolemmal depolarization. The local increase in Cai due to S-CaOs is equivalent to that induced by an AP. Heterogeneity of diastolic Cai caused by asynchronous S-CaOs among cells within myocardial tissue leads to heterogeneous myofilament activation, the summation of which produces a Ca(2+)-dependent component to diastolic tone. The local increases in Cai due to S-CaOs also cause oscillatory sarcolemmal depolarizations due to Ca2+ modulation of the Na/Ca exchanger and of non-specific cation channels. Thus, inhomogeneous levels of diastolic Cai may lead to heterogeneity in cell coupling and thus may also affect the impulse conduction. The magnitude of the S-CaOs induced diastolic tonus and membrane depolarization varies with the extent to which S-CaOs are synchronized; partially synchronized S-CaOs following an AP induced SR Ca2+ release produce an aftercontraction and after depolarization. When local S-CaOs is sufficiently synchronized within the cell the resultant depolarization summates and can be sufficient to trigger spontaneous AP. Inhomogeneity of diastolic SR Ca2+ loading and sarcomere lengths within individual cardiac cells due to S-CaOs leads to inhomogeneous systolic Cai levels and sarcomere length inhomogeneities in response a subsequent AP; this heterogeneity compromises the systolic contraction amplitude. Heterogeneity of systolic Cai among cells due to diastolic S-CaOs also leads to heterogeneity of AP repolarization times, due, to heterogeneous Cai modulation of the Na/Ca exchanger, the non-specific cation channel and of the L type sarcolemmal Ca2+ channel. S-CaOs occurrence during a long AP plateau may also modulate the removal of voltage inactivation of L type Ca2+ channels and affect the likelihood of the occurrence of "early after depolarizations." Thus, as a single entity, S-CaOs may be implicated in diverse manifestations of heart failure--impaired systolic performance, increased diastolic tonus and an increased probability for the occurrence of arrhythmias.
Animals↗
Effect of U-50,488H on the contractile response of cardiomyopathic hamster ventricular myocytes.
We examined the effects of a selective kappa opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca2+] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P less than or equal to 0.05). There was a concentration-dependent effect of U-50,488H (0.1-20 microM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P less than or equal to 0.001). In cells loaded with the Ca2+ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Functional implications of spontaneous sarcoplasmic reticulum Ca2+ release in the heart.
The Cai oscillation generated by the sarcoplasmic reticulum in response to an action potential occurs relatively synchronously within and among cells. The sarcoplasmic reticulum can also generate spontaneous Cai oscillations (S-CaOs), ie, not triggered by sarcolemmal depolarisation. The local increase in Cai due to S-CaOs is equivalent to that induced by an action potential. Heterogeneity of diastolic Cai among cells within myocardial tissue caused by asynchronous S-CaOs leads to heterogeneous myofilament activation, the summation of which produces a Ca2+ dependent component to diastolic tone. The local increases in Cai due to S-CaOs also cause oscillatory sarcolemmal depolarisations due to Ca2+ modulation of the Na-Ca exchanger and of non-specific cation channels. When local S-CaOs within a myocardial cell is sufficiently synchronised the resultant depolarisation summates and can be sufficient to trigger a spontaneous action potential. Inhomogeneous levels of diastolic Cai among cells may lead to heterogeneity in cell coupling and thus may also affect the impulse conduction in myocardial tissue. The magnitude of the S-CaOs induced diastolic tonus and depolarisation varies with the extent to which S-CaOs are synchronised; partially synchronised S-CaOs following an action potential induced Ca2+ release produce an aftercontraction and afterdepolarisation. Inhomogeneity of diastolic sarcoplasmic reticulum Ca2+ loading and sarcomere lengths within individual cardiac cells due to S-CaOs leads to inhomogeneous systolic Cai levels and sarcomere inhomogeneities in response to a subsequent action potential, which compromise the systolic contraction amplitude. Heterogeneity of systolic Cai among cells due to diastolic S-CaOs also leads to heterogeneity of action potential repolarisation times, due to heterogeneous Cai modulation of the Na-Ca exchanger, the non-specific cation channel, the L type Ca2+ channel and, depending upon species, Ca2+ activated K+ channels. S-CaOs occurrence during a long action potential plateau may also modulate the removal of voltage inactivation of L type Ca2+ channels, and affects the likelihood of the occurrence of "early afterdepolarisations." Thus, as a single entity, S-CaOs may be implicated in diverse manifestation of heart failure--impaired systolic performance, increased diastolic tonus, and an increased probability for the occurrence of arrhythmias.
Calcium↗
Excitation-contraction coupling in the heart: the state of the question.
Recent developments have led to great progress toward determining the mechanism by which calcium is released from the sarcoplasmic reticulum in the heart. The data support the notion of calcium-induced calcium release via a calcium-sensitive release channel. Calcium release channels have been isolated and cloned. This situation creates a paradox, as it has also been found that calcium release is smoothly graded and closely responsive to sarcolemmal membrane potential, properties that would not be expected of calcium-induced calcium release, which has intrinsic positive feedback. There is, therefore, no quantitative understanding of how the properties of the calcium release channel can lead to the macroscopic physiology of the whole cell. This problem could, in principle, be solved by various schemes involving heterogeneity at the ultrastructural level. The simplest of these require only that the sarcolemmal calcium channel be located in close proximity to one or more sarcoplasmic reticulum release channels. Theoretical modeling shows that such arrangements can, in fact, resolve the positive feedback paradox. An agenda is proposed for future studies required in order to reach a specific, quantitative understanding of the functioning of calcium-induced calcium release.
Animals↗
Interaction between nutrition and aging: a summary of effects on the cardiovascular system.
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Adult↗
Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.
1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Contractile response of individual cardiac myocytes to norepinephrine declines with senescence.
The present study utilized individual isolated left ventricular cardiac myocytes from hearts of animals of a broad age range to evaluate the response to norepinephrine and to other stimuli that augment myocardial cell contractile performance. During electrical stimulation before drugs neither the amplitude nor the velocity of shortening normalized for resting cell length differed among cells isolated from 2-, 6- to 8-, or 24-mo-old animals. Norepinephrine augmented twitch amplitude and velocity about fourfold in cells from 2-mo-old hearts but only by 2.5-fold in cells from 24-mo-old hearts (age effect, P less than 0.001). In contrast, the contractile response to increases in bathing [Ca2+] or to the addition of the calcium channel agonist BAY K 8644 or of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT cAMP) did not vary with age. These results indicate that the age-associated contractile deficit during beta-adrenergic stimulation is specific to the beta-adrenergic pathway and an age-associated deficit in the net production of cAMP. This can be attributed to a diminished cardiac myocyte response to beta-adrenergic agonists, in contrast to modulation of the beta-adrenergic response by other receptor agonists, which are present in intact tissue but absent under the conditions of the present study.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Novel technique to load indo-1 free acid into single adult cardiac myocytes to assess cytosolic Ca2+.
This report introduces a novel method to load indo-1 "free acid" selectively into the cytosol of cardiac myocytes, presumably by diffusion through momentarily permeable gap junction sites during mechanical dissociation after low-Ca2+ collagenase treatment. Calibration of indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence (AF) from time-matched non-indo-loaded cells, taking into account apparent changes in cell AF due to indo-1. There is wide variation in the degree of uncertainty of individual intracellular Ca2+ concentration ([Ca2+]i) determinations among cells, related principally to differences in cellular indo-1 content, to nonlinear aspects of the [Ca2+]-to-fluorescence ratio relationship, and to the uncertainty in the AF subtraction. Consequently, a quantitative estimate of uncertainty also may be employed in formulating weighted estimates of cytosolic [Ca2+]i. The following [Ca2+]i values in rat ventricular cells (nM; in 1 mM bathing extracellular Ca2+ concentration, 25 degrees C) are given as weighted means +/- 95% confidence intervals (unweighted values in parentheses): 138 +/- 5 (136 +/- 6, n = 44) in quiescent cells, 435 +/- 74 (482 +/- 76, n = 43) at the [Ca2+]i-transient peak during 0.5 Hz steady-state stimulation, and 760 +/- 124 (1,027 +/- 250, n = 42) at the [Ca2+]i-transient peak, postrest. Moreover, these peak [Ca2+]i values fall near the steepest portion of the force-Ca2+ curve (from intact cardiac muscle), consistent with sensitive inotropic regulation and maximal contractile reserve.
Acids↗
Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1.
This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Age-related decline in left ventricular filling at rest and exercise.
To determine whether the age-associated decline in resting left ventricular diastolic filling persists during aerobic exercise, rest and bicycle exercise filling indexes were measured from gated radionuclide blood pool scans in 88 healthy men aged 22-82 yr. To evaluate the effect of physical conditioning status on these age-related changes, a subset of the subjects consisted of endurance-trained senior athletes with a maximal O2 consumption of 50.5 +/- 5 compared with 32.6 +/- 7 ml.kg-1 x min-1 in age-matched controls. The contribution of beta-adrenergic stimulation to exercise-induced changes in filling was also evaluated by the administration of intravenous propranolol to another subset before testing. Peak filling rate increased progressively at all ages with increasing exercise work loads. The peak filling rates at rest, 50% maximal exercise, and maximum exercise inversely correlated with age (r = -0.64, -0.53, -0.64, respectively). Rest and exercise filling indexes in senior athletes were similar to those of sedentary older subjects. Propranolol decreased exercise peak filling rates in young (37.2 +/- 7.5 yr) but not in older (62.1 +/- 6 yr) subjects. Therefore, filling rates increase with exercise in both young and older healthy men, but age differences persist at comparable relative work loads. This decline is not secondary to a decline in physical conditioning status but appears to be related to a decrease in beta-adrenergic responsiveness in older individuals.
Adrenergic beta-Antagonists↗
Kappa and delta opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from an intracellular pool in myocytes and neurons.
We investigated the effects of mu, delta, and kappa opioid receptor stimulation on the contractile properties and cytosolic Ca2+ (Cai) of adult rat left ventricular myocytes. Cells were field-stimulated at 1 Hz in 1.5 mM bathing Ca2+ at 23 degrees C. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10(-5) M) had no effect on the twitch. The delta-agonists methionine enkephalin and leucine enkephalin (10(-10) to 10(-6) M) and the kappa-agonist (trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclo-hexyl]- benzeneacetamide)methanesulfonate hydrate (U-50,488H; 10(-7) to 2 x 10(-5) M) had a concentration-dependent negative inotropic action. The sustained decrease in twitch amplitude due to U-50,488H was preceded by a transient increase in contraction. The effects of delta- and kappa-receptor stimulation were antagonized by naloxone and (-)-N-(3-furyl-methyl)-alpha-normetazocine methanesulfonate, respectively. In myocytes loaded with the Ca2+ probe indo-1, the effects of leucine enkephalin (10(-8) M) and U-50,488H (10(-5) M) on the twitch were associated with similar directional changes in the Cai transient. Myofilament responsiveness to Ca2+ was assessed by the relation between twitch amplitude and systolic indo-1 transient. Leucine enkephalin (10(-8) M) had no effect, whereas U-50,488H (10(-5) M) increased myofilament responsiveness to Ca2+. We subsequently tested the hypothesis that delta and kappa opioid receptor stimulation may cause sarcoplasmic reticulum Ca2+ depletion. The sarcoplasmic reticulum Ca2+ content in myocytes and in a caffeine-sensitive intracellular Ca2+ store in neurons was probed in the absence of electrical stimulation via the rapid addition of a high concentration of caffeine from a patch pipette above the cell. U-50,488H and leucine enkephalin slowly increased Cai or caused Cai oscillations and eventually abolished the caffeine-triggered Cai transient. These effects occurred in both myocytes and neuroblastoma-2a cells. In cardiac myocyte suspensions U-50,488H and leucine enkephalin both caused a rapid and sustained increase in inositol 1,4,5-trisphosphate. Thus, delta and kappa but not mu opioids have a negative inotropic action due to a decreased Cai transient. The decreased twitch amplitude due to kappa-receptor stimulation is preceded by a transient increase in contractility, and it occurs despite an enhanced myofilament responsiveness to Ca2+. The effects of delta and kappa opioids appear coupled to phosphatidylinositol turnover and, at least in part, may be due to sarcoplasmic reticulum Ca2+ depletion.(ABSTRACT TRUNCATED AT 400 WORDS)
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na(+)-Ca2+ exchange and not by its antioxidant effects.
Myocardial reoxygenation injury may be attenuated by oxygen free radical scavengers, arguing for a role of oxygen radicals in this process. To determine whether free radical scavengers affect reoxygenation injury in isolated cardiac myocytes, resting rat ventricular myocytes were exposed to hypoxic (PO2 less than 0.02 mm Hg) glucose-free buffer alone (n = 50) or with the addition of the oxygen radical scavengers 1,3-dimethyl-2-thiourea (DMTU, 25 mM, n = 46), human recombinant superoxide dismutase (SOD, 1,000 units/ml, n = 40), or the combination of these agents (n = 41). All cells responded by undergoing contracture to a rigor form. Hypoxia was then continued for a second period (T2), the duration of which correlates inversely with survival. After reoxygenation, cells either retained their rectangular shape (survival) or hypercontracted to a rounded form (death). For the group of cells with a T2 period greater than 30 minutes, no cell exposed to buffer alone (n = 20) or to SOD (n = 16) survived, in contrast to 15 of 24 (63%) cells exposed to DMTU. The addition of SOD to DMTU offered no advantage to DMTU alone. The protective effect of DMTU was not observed when it was added at reoxygenation, suggesting that this agent has an important effect during the hypoxic period when intracellular Ca2+ is known to rise, most likely because of the reversal of Na(+)-Ca2+ exchange. Therefore, the effects of DMTU on Ca2+ regulation (indexed by indo-1 fluorescence) during hypoxia were studied. DMTU significantly blunted the [Ca2+] rise during the hypoxic period.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of the sarcomeric actin isogenes in the rat heart with development and senescence.
Sarcomeric actin genes, alpha-cardiac and alpha-skeletal, are coexpressed in neonatal rodent hearts and are regulated in response to hormonal and hemodynamic stimuli; however, their precise developmental pattern of expression has not been determined, and it is unknown whether they are coexpressed during senescence. We have, therefore, investigated the accumulation of sarcomeric actin transcripts in rat heart during fetal and postnatal development and with senescence by two different techniques: primer extension analysis with an oligonucleotide common to both sarcomeric actins and RNA hybridization with specific cardiac alpha-actin cRNA probes. We found that at 17-19 days in utero both isogenes are coexpressed and alpha-skeletal actin mRNAs represent 28.0 +/- 0.8% of the sarcomeric actin mRNA total. Skeletal actin mRNAs increase to 40% of the total 1 week after birth (NS, p = 0.15), remain constant for 3 weeks, and decrease to less than 20% of the total in ventricles and atria of 1-month-old rats. The alpha-skeletal actin transcripts further decline to less than 5% of the total at 2 months of age and do not reaccumulate in senescent animals. There was no significant difference between male and female rat ventricles. By comparison with the known accumulations of alpha- and beta-myosin heavy chain mRNAs, our results demonstrate that whatever the developmental stage the kinetics of expression for the sarcomeric myosin and actin multigene families are independent.
Actins↗
Novel diazinone derivatives separate myofilament Ca2+ sensitization and phosphodiesterase III inhibitory effects in guinea pig myocardium.
The inotropic state of the myocardium can be enhanced via an increase in cell Ca2+ loading or in myofilament responsiveness to Ca2+. Although different pharmacological agents combine these properties, no presently available drug acts predominantly as a myofilament sensitizer in situ. We have investigated the effects and the mechanism of action of novel diazinone derivatives, EMD 54622, EMD 53998, and EMD 54650 (developed by E. Merck, Darmstadt), on guinea pig myocardial preparations. Force- and ATPase-pCa relations in skinned fibers show differing potencies of these agents on myofilament sensitization: EMD 54622 greater than EMD 53998 much greater than EMD 54650. This is in contrast to their relative potencies to inhibit isolated myocardial phosphodiesterase III: EMD 54650 greater than EMD 53998 greater than EMD 54622. In isolated hearts studied at constant coronary flow, each of the three diazinone derivatives had a positive inotropic effect. In enzymatically dissociated left ventricular myocytes loaded with the Ca2+ probe indo-1, the positive inotropic effect of EMD 54622 occurred with no change in the amplitude of the cytosolic [Ca2+] (Cai) transient. In contrast, both EMD 53998 and EMD 54650 enhanced Cai transient and twitch contraction amplitudes. Length-indo-1 fluorescence relations were analyzed to determine the effects of the three substances on myofilament responsiveness to Ca2+. EMD 54622 enhanced and EMD 54650 had no effect on myofilament responsiveness to Ca2+. Less uniform results were obtained with EMD 53998 (in two of five cells the myofilament responsiveness to Ca2+ was increased, whereas in three other cells it was unaltered). Our results indicate that structural changes in the diazinone molecule shift the mechanism of action for the positive inotropic effect of the diazinone derivatives in the intact cell from a predominant myofilament sensitization (EMD 54622) to an enhancement in cell Ca2+ loading and an augmentation in the Cai transient (EMD 54650).
Actin Cytoskeleton↗
Dependence of hypoxic cellular calcium loading on Na(+)-Ca2+ exchange.
Na(+)-Ca2+ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+ loading and damage in the heart. Despite the fact that both [Na+]i and [Ca2+]i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]i occurring during hypoxia is linked to the rise in [Na+]i via Na(+)-Ca2+ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na(+)-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (PO2 less than 0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]i fell. The increase in both [Na+]i and [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na(+)-free buffer. These experiments suggested that hypoxic Ca2+ loading is linked to a rise in Na+i via Na(+)-Ca2+ exchange. To show that Na(+)-Ca2+ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+ was abruptly removed after contracture occurred. The sudden removal of buffer Na+ would be expected to stimulate cell Ca2+ entry via Na(+)-Ca2+ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+ loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na(+)-containing buffer (144 mM NaCl, n = 38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na(+)-free buffer (144 mM choline chloride, n = 17) hypercontracted at reoxygenation (p less than 0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+ current. R 56865 blunted the rise in [Na+]i typically seen after the onset of rigor, suggesting that Na+ entry may occur, in part, through voltage-gated Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia.
Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+ quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]m averaged 94 +/- 3 nM (n = 16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124 +/- 4 nM, n = 12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]m and [Ca2+]c rose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]m showed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]m or [Ca2+]c remained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]m remained higher in cells that hypercontracted (305 +/- 36 nM) than in cells that recovered (138 +/- 9 nM, p less than 0.05), whereas [Ca2+]c did not differ between the two groups (156 +/- 10 versus 128 +/- 10 nM, respectively; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Different effects of alpha- and beta-adrenergic stimulation on cytosolic pH and myofilament responsiveness to Ca2+ in cardiac myocytes.
alpha-Adrenergic stimulation (alpha-AS) and beta-adrenergic stimulation (beta-AS) of the myocardium are associated respectively with an increase and a decrease in myofilament responsiveness to Ca2+. We hypothesized that changes in cytosolic pH (pH(i)) may modulate these opposite actions of alpha-AS and beta-AS. The effects of alpha-AS (50 microM phenylephrine and 1 microM nadolol) and beta-AS (0.05 microM isoproterenol) on contraction and either cytosolic Ca2+ (Cai) or pH(i) were assessed in adult rat ventricular myocytes bathed in bicarbonate buffer (pH 7.36 +/- 0.05). In cells loaded with the ester derivative (AM form) of indo-1, the 410/490-nm ratio of emitted fluorescence indexed Cai. Myofilament responsiveness to Ca2+ was assessed by the relaxation phase of the length-indo-1 fluorescence relation during a twitch. alpha-AS and beta-AS shifted this relation in opposite directions, indicating that alpha-AS increased and beta-AS decreased myofilament responsiveness to Ca2+. In addition, the positive inotropic action of alpha-AS was associated with an increased Cai transient amplitude in 50% of the myocytes (n = 12), whereas beta-AS always increased Cai (n = 5). In cells loaded with the fluorescent pH(i) probe SNARF-1 AM, the emitted 590/640-nm fluorescence is a measure of pH(i). The effect of alpha-AS on the extent of cell shortening during the twitch (ES) was expressed as the percentage of resting cell length. Both ES and pH(i) were assessed in myocytes bathed in 1.5 mM [Ca2+] and stimulated at 0.5 Hz (control ES, 7.4 +/- 1.5%; control pH(i), 7.11 +/- 0.05; n = 10). alpha-AS enhanced both ES (delta ES, 1.8 +/- 0.6%; p less than 0.05) and pH(i) (delta pH(i), 0.06 +/- 0.01; p less than 0.005), and there was a significant correlation between delta ES and delta pH(i) (r = 0.76, p less than 0.05). A similar effect of alpha-AS on pH(i) was observed in the absence of electrical stimulation (n = 8). The alpha-AS-induced enhancement of ES and pH(i) was abolished by 10 microM ethylisopropylamiloride, a Na(+)-H+ exchange inhibitor (n = 7). In additional experiments, myocytes were preincubated either with 0.2 microM 4 beta-phorbol 12-myristate 13-acetate (n = 8) or with 5 nM staurosporine (n = 8), which have been shown to downregulate and inhibit Ca(2+)-activated phospholipid-dependent protein kinase C, respectively. In either group, alpha-AS had no effect on pH(i) and decreased ES to approximately 60% of control.(ABSTRACT TRUNCATED AT 400 WORDS)
Actin Cytoskeleton↗