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Alterations in calcium homeostasis, energy metabolism, and membrane integrity during metabolic inhibition in cultured neonatal rat ventricular myocytes
Cardiol
21 (Supplement
J Mol
Cell
232
ALTERATIONS IN CALCIUM HOMEOSTASIS, ENERGY METABOLISM, AND MEMBRANE INTEGRITY DURING METABOLIC INHIBITION IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES . A.C. MORRIS, H.K. HAGLER, L.M. BUJA. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. Cell injury resulting from myocardial ischemia is associated with altered elemental profiles, impaired energy metabolism, and sarcolemmal degradation. In this study we have used a cultured neonatal rat ventricular myocyte (M), model to examine the effects of 1 or 2 hours of metabolic inhibition with iodoacetate (IAA, 30 PM) on cellular ionic calcium [Ca2+]i levels and distribution using fura 2, total elemental profiles using x-ray microanalysis (EPMA), and ATP levels. For studies of [Ca2+]i, M were grown on glass coverslips for 3 days, treated with M-199 containing 3.0 PM fura 2/AM for 30 min, equilibrated in fura-free M-199 for 60 min, placed in a Sykes Moore chamber on the heated stage of an inverted microscope equipped with a Fluoroplex 111-1000 microspectrofluorometer and image analysis system. [Ca2+]i was determined by alternating 340 and 380nm excitation with a ten segment chopper wheel and monitoring emission with a photomultiplier tube and a low light video camera. For studies of total elemental content by subcellular compartment M were prepared as previously reported, (Buja et.al., Lab Invest 53:397-412, 1985). Under control conditions M displayed [Ca2+]i transients associated with spontaneous contractions ranging from 200 nM at relaxation to 900 nM with contraction. After Ihr of superfusion with IAA spontaneous contractions had ceased and [Ca2+]i was stable at 150 nM. 2 hr of IAA treatment resulted in contracture and sarcolemmal blebbing accompanied by elevated [Ca2+]i to 5 FM. Fluorescence image analysis confirmed spatial variation in the magnitude of [Ca2+] elevation in individual cells. EPMA showed progressive increases in Ca in cytoplasm and mitochondria between 1 and 2 hrs of IAA treatment. ATP levels in M treated with IAA for 1 or 2 hr were reduced by 30% and 75% respectively. Thus, prolonged metabolic inhibition with IAA in this model results in pronounced depression of ATP levels which accompany elevation in total and ionic [Ca2+], sarcolemmal blebbing, and cell injury.
233
CHANGES SFBAPTA
England. 02115.
IN NMR.
INTRACELLULAR
II) (1989)
CALCIUM
IN
ISCHEMIC
FERRET
HEARTS
EVIDENCED
BY
JW Tsao* and PG Morris, Department of Biochemistry, Cambridge University, Cambridge CB2 lQW, *Present address: Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA
Stndies of cytosolic calcium levels during ischemia and reperfusion in intact hearts are of interest as these conditions can only be mimicked in celhdaz preparations. In this study, Langendorff-perfused ferret hearts were loaded with the lgF NMR calcium indicator, 5,S-difluoro-l,2-bis(o-aminophenoxy)ethane-N~,~~-te~cetic acid (SFBAPTA) and subjected to simulated ischemia of both short (20-30 min) and long (>30 min) duration. NMR data were collected in 45 set intervals. When analyzed in 3 min blocks, an immediate fall in overall intracellular calcium was apparent, from 890 + 130 nM (mean f SEM) to 270 & 20 nM, complete by 15 min (n=6,O.C@l
234
IN SPONTANEOUS INFLUENCES OF EXTRACELLULAR Ca ON INTRACELLULAR Ca++ AND CONTRACTILITY REATTNC. HEART CELLS. H.Ishida. H.Seguchi. K.Hosono*. U.Eki. N.Aosaki. F.Ohsuzu. of Medicine, latiobal Defense .Medical. College. *Mitsukoshi H.Nakamura. 1st Dept. Health & Welfare Foundations, Japan. Although it is well known that the alteration of extracellular Ca concentration ([Calo) changes contractility, the influence of [Ca]o on intracellular Ca++ ([Ca”li) We established new method that could measure simultaneously is less understood. contractile motion with video motion detector and [Ca+*]i transient with Indo-l using microscope-fluorescent system. We examined that influences of [Ca]o on amplitude of motion, beating rate and [Ca”] in spontaneous beating of cultured heart cells. l P
21 (Supplement
J Mol
Cell
232
ALTERATIONS IN CALCIUM HOMEOSTASIS, ENERGY METABOLISM, AND MEMBRANE INTEGRITY DURING METABOLIC INHIBITION IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES . A.C. MORRIS, H.K. HAGLER, L.M. BUJA. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. Cell injury resulting from myocardial ischemia is associated with altered elemental profiles, impaired energy metabolism, and sarcolemmal degradation. In this study we have used a cultured neonatal rat ventricular myocyte (M), model to examine the effects of 1 or 2 hours of metabolic inhibition with iodoacetate (IAA, 30 PM) on cellular ionic calcium [Ca2+]i levels and distribution using fura 2, total elemental profiles using x-ray microanalysis (EPMA), and ATP levels. For studies of [Ca2+]i, M were grown on glass coverslips for 3 days, treated with M-199 containing 3.0 PM fura 2/AM for 30 min, equilibrated in fura-free M-199 for 60 min, placed in a Sykes Moore chamber on the heated stage of an inverted microscope equipped with a Fluoroplex 111-1000 microspectrofluorometer and image analysis system. [Ca2+]i was determined by alternating 340 and 380nm excitation with a ten segment chopper wheel and monitoring emission with a photomultiplier tube and a low light video camera. For studies of total elemental content by subcellular compartment M were prepared as previously reported, (Buja et.al., Lab Invest 53:397-412, 1985). Under control conditions M displayed [Ca2+]i transients associated with spontaneous contractions ranging from 200 nM at relaxation to 900 nM with contraction. After Ihr of superfusion with IAA spontaneous contractions had ceased and [Ca2+]i was stable at 150 nM. 2 hr of IAA treatment resulted in contracture and sarcolemmal blebbing accompanied by elevated [Ca2+]i to 5 FM. Fluorescence image analysis confirmed spatial variation in the magnitude of [Ca2+] elevation in individual cells. EPMA showed progressive increases in Ca in cytoplasm and mitochondria between 1 and 2 hrs of IAA treatment. ATP levels in M treated with IAA for 1 or 2 hr were reduced by 30% and 75% respectively. Thus, prolonged metabolic inhibition with IAA in this model results in pronounced depression of ATP levels which accompany elevation in total and ionic [Ca2+], sarcolemmal blebbing, and cell injury.
233
CHANGES SFBAPTA
England. 02115.
IN NMR.
INTRACELLULAR
II) (1989)
CALCIUM
IN
ISCHEMIC
FERRET
HEARTS
EVIDENCED
BY
JW Tsao* and PG Morris, Department of Biochemistry, Cambridge University, Cambridge CB2 lQW, *Present address: Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA
Stndies of cytosolic calcium levels during ischemia and reperfusion in intact hearts are of interest as these conditions can only be mimicked in celhdaz preparations. In this study, Langendorff-perfused ferret hearts were loaded with the lgF NMR calcium indicator, 5,S-difluoro-l,2-bis(o-aminophenoxy)ethane-N~,~~-te~cetic acid (SFBAPTA) and subjected to simulated ischemia of both short (20-30 min) and long (>30 min) duration. NMR data were collected in 45 set intervals. When analyzed in 3 min blocks, an immediate fall in overall intracellular calcium was apparent, from 890 + 130 nM (mean f SEM) to 270 & 20 nM, complete by 15 min (n=6,O.C@l
234
IN SPONTANEOUS INFLUENCES OF EXTRACELLULAR Ca ON INTRACELLULAR Ca++ AND CONTRACTILITY REATTNC. HEART CELLS. H.Ishida. H.Seguchi. K.Hosono*. U.Eki. N.Aosaki. F.Ohsuzu. of Medicine, latiobal Defense .Medical. College. *Mitsukoshi H.Nakamura. 1st Dept. Health & Welfare Foundations, Japan. Although it is well known that the alteration of extracellular Ca concentration ([Calo) changes contractility, the influence of [Ca]o on intracellular Ca++ ([Ca”li) We established new method that could measure simultaneously is less understood. contractile motion with video motion detector and [Ca+*]i transient with Indo-l using microscope-fluorescent system. We examined that influences of [Ca]o on amplitude of motion, beating rate and [Ca”] in spontaneous beating of cultured heart cells. l P