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W H Barry

Publications and source records attributed to W H Barry.

At least 55 records · Page 3Linked to original sources
PubMed1995

The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5' nontranslated region.

We report the construction of chimeric coxsackievirus B3 (CVB3) strains in which sequences of an infectious cDNA copy of a noncardiovirulent CVB3 genome were replaced by the homologous sequences from a cardiovirulent CVB3 genome to identify which of 10 predicted genetic sites determine cardiovirulence. Cardiovirulent phenotype expression was consistently linked to nucleotide 234 (U in cardiovirulent CVB3 and C in avirulent CVB3) in the 5' nontranslated region. Reconstructions of the parental noncardiovirulent CVB3 genome from chimeras restored the noncardiovirulent phenotype when tested in mice. Inoculation of severe combined immunodeficient (scid) mice with the noncardiovirulent CVB3 strain resulted in massive cardiomyocyte necrosis in all animals. Sequence analysis of viral genomes isolated from twelve scid mouse hearts showed that only nucleotide position 234 was different (a C-->U transition) from that in the input parental noncardiovirulent CVB3 genome. Higher-order RNA structures predicted by two different algorithms did not demonstrate an obvious local effect caused by the C-->U change at nucleotide 234. Initial studies of parental and chimeric CVB3 replication in primary cultures of fetal murine heart fibroblasts and in adult murine cardiac myocytes demonstrated that viral RNA transcriptional efficiency is approximately 10-fold lower for noncardiovirulent CVB3 than for cardiovirulent CVB3. CVB3 did not shut off protein synthesis in murine cardiac fibroblasts, nor were levels of viral protein synthesis significantly different as a function of viral phenotype. Taken together, these data support a significant role for determination of the CVB3 cardiovirulence phenotype by nucleotide 234 in the 5' nontranslated region, possibly via a transcriptional mechanism.

Animals↗
PubMed1995

Method for isolation of human ventricular myocytes from single endocardial and epicardial biopsies.

The study of adult human ventricular cells has been limited by tissue availability. In this study we describe techniques for the isolation of Ca(2+)-tolerant adult human ventricular cells from both transvenous endomyocardial and epicardial biopsies. Ca(2+)-tolerant cells were obtained from 80% of the biopsies processed. Although the yield of Ca(2+)-tolerant myocytes from either type of biopsy was low (1-5%), myocytes with normal resting potentials and action potentials can be obtained from single biopsy specimens, providing a source of normal human myocytes for electrophysiological study. Resting potentials (Vrest) were recorded in 41 isolated right ventricular endomyocardial cells at 37 degrees C. Sixteen cells were depolarized (Vrest = -26 +/- 13 mV), and 25 cells had normal resting potentials (Vrest = -84 +/- 6 mV). Action potentials were recorded in 16 cells. At a pacing cycle length of 1 s, 4 cells had prolonged action potential duration at 90% (APD90, 718 +/- 26 ms) and 10 cells had normal APD90 (381 +/- 94 ms) compared with those recorded from intact right ventricular septal trabeculae from explanted hearts. Voltage-clamp studies of isolated human ventricular myocytes obtained from these biopsies document the presence of currents previously reported from cells isolated from explanted hearts.

Animals↗
PubMed1995

Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes.

We tested the hypothesis that glycolytic inhibition by 2-deoxyglucose causes greater impairment of diastolic relaxation and intracellular calcium handling in well-oxygenated hypertrophied adult rat myocytes compared with control myocytes. We simultaneously measured cell motion and intracellular free calcium concentration ([Ca2+]i) with indo-1 in isolated paced myocytes from aortic-banded rats and sham-operated rats. There was no difference in either the end-diastolic or peak-systolic [Ca2+]i between control and hypertrophied myocytes (97 +/- 18 vs. 105 +/- 15 nM, 467 +/- 92 vs. 556 +/- 67 nM, respectively). Myocytes were first superfused with oxygenated Hepes-buffered solution containing 1.2 mM CaCl2, 5.6 mM glucose, and 5 mM acetate, and paced at 3 Hz at 36 degrees C. Exposure to 20 mM 2-deoxyglucose as substitution of glucose for 15 min caused an upward shift of end-diastolic cell position in both control (n = 5) and hypertrophied myocytes (n = 10) (P < 0.001 vs. baseline), indicating an impaired extent of relaxation. Hypertrophied myocytes, however, showed a greater upward shift in end-diastolic cell position and slowing of relaxation compared with control myocytes (delta 144 +/- 28 vs. 55 +/- 15% of baseline diastolic position, P < 0.02). Exposure to 2-deoxyglucose increased end-diastolic [Ca2+]i in both groups (P < 0.001 vs. baseline), but there was no difference between hypertrophied and control myocytes (218 +/- 38 vs. 183 +/- 29 nM, respectively). The effects of 2-deoxyglucose were corroborated in isolated oxygenated perfused hearts in which glycolytic inhibition which caused severe elevation of isovolumic diastolic pressure and prolongation of relaxation in the hypertrophied hearts compared with controls. In summary, the inhibition of the glycolytic pathway impairs diastolic relaxation to a greater extent in hypertrophied myocytes than in control myocytes even in well-oxygenated conditions. The severe impairment of diastolic relaxation induced by 2-deoxyglucose in hypertrophied myocytes compared with control myocytes cannot be explained by greater diastolic Ca2+ overload, which implicates an increase in myofilament Ca(2+)-responsiveness as a possible mechanism.

Adenosine Triphosphate↗
PubMed1995

Identification of a contractile function for renal medullary interstitial cells.

Renomedullary interstitial cells (RMIC) are unique to the renal medulla. By virtue of their anatomic location and arrangement, RMIC may hinder axial dissipation of the concentration gradient, thereby aiding urinary concentration. A more active role in urinary concentration has been postulated on the basis of speculations about RMIC contractile potential, however, RMIC contraction has not been investigated. To determine if these cells are contractile, cultured rat RMIC were exposed to endothelin-1 (ET-1), a potent vasoconstrictor which binds to RMIC, and examined using video microscopy. ET-1 (as low as 10 pM) caused a slowly developing and dose-dependent reduction in RMIC surface area. ET-1 markedly increased the number and intensity of F-actin microfilament staining. ET-1-induced RMIC contraction was not altered by nifedipine, was partially reduced by nickel, and was completely inhibited by H7, indicating that ET-1 action is mediated by protein kinase C and is partially dependent upon receptor-operated calcium channels. The ET-1 effect does not involve nitric oxide since NG-monomethyl-L-arginine did not alter ET-1-induced RMIC contraction; in addition, ET-1 had only a minor effect on cGMP levels and no effect on nitrite production. PGE2 acts in an autocrine manner to dampen ET action since indomethacin potentiates, while PGE2 inhibits, ET-1-induced RMIC contraction. The contractile response is not unique to ET-1 since vasopressin also reduces RMIC surface area and increases F-actin microfiliment staining. These studies demonstrate that RMIC in culture are contractile. The possibility is raised that contraction of RMIC plays a role in modifying urinary concentration as well as regulation of other renal medullary functions.

Animals↗
PubMed1995

Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes.

OBJECTIVE: The aim was to characterise the effects of angiotensin II on Na+/H+ exchange in adult ventricular myocytes. METHODS: Intracellular pH (pHi) was continuously measured with the fluorescent pH indicator, SNARF-1, in single resting myocytes obtained from adult rabbits by enzymatic dissociation. In some experiments cells were electrically paced to elicit contractions. All experiments were performed at 36 degrees C in HEPES buffered solution containing no added CO2 or HCO3- (pHo 7.4). RESULTS: Rapid application of angiotensin II caused pHi to rise. The initial rate of rise and initial net H+ efflux responded to angiotensin II in a concentration dependent manner, EC50 = 7.8. Buffering of cytosolic calcium with the calcium chelator BAPTA did not affect the initial net H+ efflux elicited by 1 microM angiotensin II. The increase in steady state pHi was blocked by inhibitors of Na+/H+ exchange, amiloride (1 mM) and EIPA (10 microM). Angiotensin II also increased the rate of pHi recovery from intracellular acidosis at pHi values above approximately 6.9. During inhibition of Na+/H+ exchange the application of angiotensin II decreased steady state pHi. This acidosis was blocked by preincubation in dextrose-free solution containing 20.0 mM 2-deoxy-D-glucose and 10 microM EIPA. The positive inotropic effect of angiotensin II was markedly suppressed by amiloride. CONCLUSIONS: Angiotensin II exerts a concentration dependent stimulatory effect on Na+/H+ exchange in adult rabbit ventricular myocytes. This effect does not appear to involve changes in cytosolic calcium. During inhibition of Na+/H+ exchange, angiotensin II causes pHi to fall, perhaps by stimulating metabolic acid production. The positive inotropic action of angiotensin II depends, in part, on stimulation of Na+/H+ exchange.

Amiloride↗
PubMed1994

Effects of angiotensin II on intracellular Ca2+ and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1.

1. Angiotensin II increases myocardial contractility in several species, including the rabbit and man. However, it is controversial whether the predominant mechanism is an increase in free cytosolic [Ca2+]i or a change in myofilament Ca2+ sensitivity. To address this question, we infused angiotensin II in isolated perfused rabbit hearts loaded with the Ca2+ indicator indo-1 AM and measured changes in beat-to-beat surface transients of the Ca2+i-sensitive 400:500 nm ratio and left ventricular contractility. The effects of angiotensin II were compared with the response to a Ca(2+)-dependent increase in the inotropic state produced by a change in the perfusate [Ca2+] from 0.9 to 3.6 nM. 2. In the isolated beating heart, an increase in perfusate [Ca2+] caused an increase in left ventricular pressure +dP/dt in association with an increase in peak systolic [Ca2+]i. Angiotensin II perfusion caused a similar increase in left ventricular +dP/dt in the absence of any increase in peak systolic [Ca2+]i. 3. To exclude any contribution of non-myocyte sources of Ca(2+)-sensitive fluorescence which may be present in the intact heart, we also compared the effects of angiotensin II and a change in superfusate [Ca2+] in collagenase-dissociated paced adult rabbit ventricular myocytes loaded with indo-1 AM. In the isolated rabbit myocytes a change in perfusate [Ca2+] from 0.9 to 3.6 mM caused an increase in peak systolic cell shortening coincident with an increase in peak systolic [Ca2+]i. In contrast, angiotensin II caused a similar increase in peak systolic cell shortening whereas there was no increase in peak systolic [Ca2+]i. There was also no change in inward Ca2+ current (ICa) in response to angiotensin II. 4. To investigate further the mechanism of the positive inotropic action of angiotensin II, its effects on intracellular pH were studied in isolated rabbit myocytes loaded with the fluorescent H+ probe SNARF 1. These experiments demonstrated that angiotensin II induced a 0.2 pH unit increase coincident with the development of a positive inotropic effect in isolated rabbit myocytes. 5. In summary, angiotensin II has a direct positive inotropic effect in beating rabbit hearts and in isolated paced rabbit myocytes. These experiments provide support for the hypothesis that the predominant mechanism is not an increase in free cytosolic Ca2+ but is due in part to an increase in myofilament Ca2+ sensitivity due to intracellular alkalosis.

Angiotensin II↗
PubMed1994

Effects of alloimmune injury on contraction and relaxation in cultured myocytes and intact cardiac allografts.

OBJECTIVES: This study was performed to determine the mechanisms by which allosensitized lymphocytes cause contractile dysfunction in cultured ventricular myocytes and to compare the effects on isolated myocytes with those observed in an intact heart preparation during allograft rejection. BACKGROUND: Allograft rejection may be associated with reversible abnormalities of both systolic and diastolic function. The immunologic mechanisms that cause ventricular dysfunction are poorly understood. METHODS: Vascularized heterotopic abdominal heart transplantation was performed in mice. Contractile function of excised allografts undergoing rejection was assessed using a Langendorff perfusion apparatus and a strain gauge. Spontaneously beating monolayers of cultured ventricular myocytes from donor strain fetal mice were exposed to the allosensitized cytotoxic T lymphocytes, and the effects on myocyte motion, intracellular calcium transients, relaxation half-time, membrane potential and myocyte lysis (chromium-51 release) were measured. RESULTS: In intact hearts, histologically mild rejection without myocyte necrosis was associated with decreased systolic function without slowing of relaxation. In cultured fetal myocytes, sensitized lymphocytes induced a progressive decrease in the amplitudes of myocyte motion and calcium transients, with cessation of beating within 40 min. Also, the diastolic membrane potential and amplitude of the action potential decreased. Relaxation half-time, as estimated by measurement of cell motion, was unchanged. The effect was allospecific and was reversible with early removal of lymphocytes from the myocyte monolayer. Pretreatment of lymphocytes with the degranulation inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene blocked both the negative inotropic effect and myocyte lysis. CONCLUSIONS: We conclude that impaired relaxation is not a prominent feature of contractile dysfunction caused directly in myocytes by alloimmune injury from cytotoxic lymphocytes. Allosensitized lymphocytes can cause reversible systolic dysfunction in myocytes by means of a direct cell-cell interaction. This effect may be in part responsible for the reversible systolic dysfunction associated with allograft rejection.

Animals↗
PubMed1994

Effect of 2,3-butanedione monoxime on myocyte resting force during prolonged metabolic inhibition.

The chemical phosphatase 2,3-butanedione monoxime (BDM) has been reported to inhibit both Ca(2+)-induced myofilament force development and rigor due to ATP depletion. However, during prolonged hypoxia in cultured ventricular myocytes BDM delays but does not prevent a marked increase in resting force. To investigate the mechanisms involved we measured the effects of BDM on intracellular Ca2+ concentration ([Ca2+]i; indo 1), force development (video motion detector), and ATP contents (luciferase assay) in cultured embryonic chick ventricular myocytes and adult rabbit ventricular myocytes subjected to prolonged metabolic inhibition with 1 mM NaCN and 20 mM 2-deoxyglucose. In the absence of metabolic inhibition, 20 mM BDM depressed force development even when [Ca2+]i was markedly elevated by exposure to zero-Na solution or 10 mM caffeine in chick cells, and 30 mM BDM completely inhibited Ca(2+)-induced force development in rabbit myocytes. During metabolic inhibition, 20 mM BDM delayed the onset of an increase in resting force (from 5.44 +/- 0.87 to 13.67 +/- 1.34 min in chick myocytes; from 19.13 +/- 2.23 to 32.43 +/- 3.30 min in rabbit myocytes, means +/- SE, n = 8-9). However, the rates of ATP depletion and rise in [Ca2+]i after metabolic inhibition were not altered by BDM. In the presence of BDM, during prolonged metabolic inhibition in both chick and rabbit myocytes, abrupt spontaneous or evoked alterations in [Ca2+]i were associated with corresponding changes in force. During the initial increase in resting force induced by metabolic inhibition, exposure to BDM caused a partial transient relaxation. We conclude that the delayed increase in resting force during metabolic inhibition in the presence of BDM is due to redevelopment of Ca2+ sensitivity of the myofilaments in the presence of an increased [Ca2+]i as a consequence of severe ATP depletion, whereas in the absence of BDM the more rapidly developing increase in resting force during metabolic inhibition is initially due to a rise in [Ca2+]i followed by development of rigor.

Adenosine Triphosphate↗
PubMed1994

Mechanisms of immune-mediated myocyte injury.

Much progress has been made in defining the mechanisms by which altered systolic and diastolic function of the heart may be produced by components of the immune system activated during allograft rejection and myocarditis and in patients with dilated cardiomyopathy. It is clear that injury of the vascular bed can occur via both humoral and cellular mediators and probably accounts for the acute alterations in ventricular compliance that occur during allograft rejection, as well as the accelerated development of graft atherosclerosis. Altered myocyte function and lysis can be produced by CTL in vitro, but the importance of this injury process in vivo remains uncertain. Other cells present in the inflammatory infiltrate can also affect myocyte function and survival. Neutrophils may cause lysis of myocytes, and cytokines produced by infiltrating macrophages and HtL may reach a sufficient concentration in the interstitial microenvironment to decrease myocyte catecholamine responsiveness and/or directly depress myocyte contractility. Humoral antibodies to myocyte cell surface antigens may cause cell damage by an antibody-dependent cytotoxic cell mechanism or by directly binding to and altering sarcolemmal receptor and/or ion channel function. Further elucidation of the extent of involvement of these different mechanisms in specific clinical settings may provide a basis for improved therapy of immune-mediated cardiac injury and dysfunction.

Animals↗
PubMed1993

ATP depletion causes a reversible decrease in Na+ pump density in cultured ventricular myocytes.

To examine factors contributing to impaired K+ homeostasis induced by prolonged but sublethal ATP depletion, we subjected cultured chick ventricular myocytes to metabolic inhibition with 20 mM 2-deoxy-D-glucose plus 1 mM NaCN for 2 h and then allowed myocytes to recover for 5 days in medium containing 6% fetal calf serum (FCS) or in hormone-supplemented serum-free medium. We measured spontaneous contractions (with a video motion detector), K+ content, K+ uptake, membrane potential, and Na+ pump density ([3H]ouabain binding). Exposure to metabolic inhibition for 2 h caused an acute decrease in Na+ pump site density [8.2 +/- 1.1 to 3.8 +/- 0.8 (SE) pmol/mg protein; n = 9, P < 0.02]. Compared with control cells (no metabolic inhibition, cultured for 5 days in serum-free medium), Na+ pump density remained depressed in cells recovered from metabolic inhibition in serum-free medium (3.0 +/- 0.7 pmol/mg), and this was associated with persistently depressed K+ uptake (54% of control), K+ content (67% of control), and membrane depolarization (-19 +/- 2 mV), a significant decrease in cell number (79% of control), and failure to resume spontaneous contractions. Exposure of cells inhibited for 2 h to culture medium containing 6% FCS resulted in a return of Na+ pump site density toward normal levels by 5 days, associated with recovery of K+ uptake and K+ content, preservation of cell number, and resumption of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Variable effects of endothelin-1 on [Ca2+]i transients, pHi, and contraction in ventricular myocytes.

We examined the effects of endothelin-1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) transients, intracellular pH (pHi), and cell contraction in both embryonic and neonatal as well as in adult ventricular myocytes. Exposure of chick ventricular myocytes to ET-1 (10 nM) significantly decreased both peak systolic and end-diastolic [Ca2+]i (from 949 +/- 43 to 628 +/- 59 nM and from 230 +/- 13 to 162 +/- 8 nM, respectively; P < 0.05, n = 12). The amplitude of cell contraction was also decreased during exposure to 10 nM ET-1 (81.7 +/- 1.2% of control, P < 0.01, n = 12). Exposure to 10 nM ET-1 slightly decreased pHi (-0.055 +/- 0.020 U; P < 0.05). Exposure of cultured neonatal rat ventricular myocytes to ET-1 (10 nM) produced similar effects. Responses of adult rabbit ventricular myocytes to ET-1 were dramatically different from those of embryonic or neonatal ventricular myocytes. Exposure to 10 nM ET-1 increased the amplitude of cell contraction to 159 +/- 32% of control (P < 0.01) without an increase in [Ca2+]i transients. ET-1 also increased pHi (+0.081 +/- 0.047 U; P < 0.01). These results indicate that ET-1 produces a negative inotropic effect by decreasing [Ca2+]i transients and induces a slight intracellular acidosis in immature ventricular myocytes. However, ET-1 causes a positive inotropic effect in adult ventricular myocytes via an intracellular alkalinization, rather than by an increase in the [Ca2+]i transient. Thus the response of myocytes to vasoactive peptides may vary with development and/or species.

Aging↗
PubMed1993

Intracellular calcium homeostasis in cardiac myocytes.

Calcium homeostasis in cardiac myocytes results from the integrated function of transsarcolemmal Ca2+ influx and efflux pathways modulated by membrane potential and from intracellular Ca2+ uptake and release caused predominantly by SR function. These processes can be importantly altered in different disease states as well as by pharmacological agents, and the resulting changes in systolic and diastolic [Ca2+]i can cause clinically significant alterations in contraction and relaxation of the heart. It may be anticipated that a rapid increase in our understanding of the pathophysiology of Ca2+ homeostasis in cardiac myocytes will be forthcoming as the powerful new tools of molecular and structural biology are used to investigate the regulation of Ca2+ transport systems.

Animals↗
PubMed1992

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1991

Lysophosphatidylcholine increases cytosolic calcium in ventricular myocytes by direct action on the sarcolemma.

Lysophosphatidylcholine (LPC) accumulates in myocardial tissues during ischemia, and has toxic effects which may contribute to the arrhythmias and relaxation abnormalities that occur during acute ischemia. These effects of LPC may be mediated in part by calcium overload. To test this hypothesis, spontaneously contracting cultured embryonic chick ventricular myocytes were superfused with various concentrations of LPC (10, 50 and 100 microM) while effects on contractile motion (video motion detector) and changes in free intracellular calcium ion concentration ([Ca2+]i indo-1 fluorescence) were determined. At concentrations greater than or equal to 10 microM, a dose-related, time-dependent effect occurred after exposure to LPC, consisting of the development of contracture and marked elevation of [Ca2+]i. LPC also produced a dose-related, time-dependent inhibition of K+ uptake, indicating there was inhibition of the Na(+)-K+ ATPase Na+ pump. However, the LPC-induced increase in [Ca2+]i was not due to Na+ overload caused by inhibition of the Na(+)-K+ ATPase Na+ pump because superfusion with a zero-Na+ solution did not prevent an increase in [Ca2+]i after LPC exposure; and the increase in [Ca2+]i after exposure to LPC occurred too rapidly to be accounted for by Na+ pump inhibition. Removal of extracellular Ca2+ prevented the rise in [Ca2+]i, after exposure to LPC but treatment with verapamil failed to inhibit the increase in [Ca2+]i induced by LPC. We conclude that LPC produces contracture due to an increase [Ca2+]i. These effects are seen at concentrations of 10 microM and greater, are not due to altered Na(+)-K+ ATPase Na+ pump or calcium channel function, and are probably related to the detergent properties of this amphiphile. There effects may account in part for myocardial dysfunction during ischemia in intact tissue.

Animals↗
PubMed1991

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+.

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.

Animals↗
PubMed1991

Mechanisms of reoxygenation injury in cultured ventricular myocytes.

To investigate factors contributing to reperfusion and reoxygenation myocardial injury, we exposed layers of cultured chick ventricular myocytes to severe hypoxia for up to 3 hours in the presence of 20 mM 2-deoxyglucose, zero glucose, and 5 mM pyruvate, and then exposed the myocytes to reoxygenation. Lactate dehydrogenase (LDH) release was moderately increased during 3 hours of hypoxia but was increased markedly during reoxygenation. Coincident changes in intracellular calcium concentration ([Ca2+]i) and cell motion were also measured during hypoxia and reoxygenation. During hypoxia, [Ca2+]i increased to more than 1 microM, and with reoxygenation, [Ca2+]i abruptly decreased slightly but remained elevated more than 1 microM. Cells developed a stable rigor after 30 minutes of hypoxia. Reoxygenation caused a marked hypercontracture within 5 minutes. Pretreatment of myocytes with either 2,3-butanedione monoxime, which inhibits Ca2(+)-dependent force development, or cyanide inhibited reoxygenation hypercontracture. LDH release after reoxygenation was also significantly reduced in the presence of 2,3-butanedione monoxime. Treatment of myocytes with superoxide dismutase and catalase during hypoxia also resulted in a decrease in LDH release during reoxygenation. We conclude that an abrupt increase in [Ca2+]i during reoxygenation does not account for reoxygenation injury. However, in the presence of elevated [Ca2+]i, reoxygenation and the resulting probable resynthesis of ATP causes [Ca2+]i-dependent myofilament crossbridge cycling, and the resulting hypercontracture contributes to myocyte damage. The generation of oxygen free radicals after reoxygenation also appears to contribute to cell injury in this system.

Adenosine Triphosphate↗
PubMed1991

Myocyte injury and contraction abnormalities produced by cytotoxic T lymphocytes.

BACKGROUND: The mechanisms by which ventricular function is altered during cardiac transplant rejection are not well understood. Therefore, an in vitro model system has been developed to facilitate investigation of lymphocyte-mediated myocyte injury. METHODS AND RESULTS: Splenic lymphoid cells were obtained from mice 8-10 days after placement of a vascularized abdominal cardiac allograft and were restimulated in vitro with irradiated donor-type splenocytes for 5 days. Cytotoxic effects of these allogenically stimulated lymphocytes on syngeneic and donor strain fetal cultured myocytes were determined by a 51Cr release assay at different lymphocyte to myocyte ratios. 51Cr release from donor strain myocytes was detectable within 1 hour of exposure, was maximal by 3-5 hours of coincubation with sensitized lymphocytes, and was allospecific. Cell injury manifest by 51Cr release was calcium dependent and was inhibited by pretreatment of lymphocytes with phorbol ester to deplete protein kinase C. Myocyte injury was also prevented by pretreatment of sensitized lymphocytes with anti-Thy 1.2 or anti-CD8 antibody plus complement but not by treatment with anti-CD4 antibody, indicating that CD8+ cytotoxic T cells are involved. Altered myocyte contractile motion preceded myocyte lysis (51Cr release), was characterized by an initial reversible decrease in amplitude of contraction, and was followed by rapid and irregular beating with eventual complete cessation of contraction. Contractile alterations induced by sensitized lymphocytes were inhibited by elimination of CD8+ cells. CONCLUSIONS: Myocyte injury can be produced by sensitized cytotoxic T lymphocytes in vitro and is calcium and protein kinase C dependent. The contractile abnormalities produced appear to be similar to those observed in cardiac transplant patients undergoing rejection, and thus this model system promises to allow investigation of the mechanisms involved.

Abdomen↗