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The ventricular-like myosin expressed in regenerating chicken muscle
J Mol Cell Cardiol21 (Supplement III) (1989) 74THE VENTRICULAR-LIKE MYOSIN EXPRESSED IN REGENERATING CHICKEN MUSCLE. Alexandre Stewart, Madhu Gupta, Blanca Camoretti-Mercado, Smilja Jakovcic and Radovan Zak. University of Chicago, Chicago, Illinois 60637, U.S.A. The anterior latissimus dorsi (ALD) muscles of chickens aged 5 weeks were cold injured with a liquid nitrogen-cooled steel rod. The area of regeneration produced by the injury was sampled for immunohistochemical analysis using a ventricular specific monoclonal antibody (CCM-18) as well as an antibody to slow myosin (HPM-7). The regenerating muscle showed strong reactivity to the ventricular-specific antibody. We are interested in determining whether this ventricular-like myosin is the same as the isoform seen in the adult ventricle. We have isolated a cDNA clone from a chicken ventricular cDNA library and northern blot analysis shows it to be ventricular specific. Preliminary sequence analysis shows that this clone shares homologous sequences with the chicken embryonic myosin heavy chain gene exons (Robbins et al., 1987). We are currently constructing a cDNA library from injured muscle so that a ventricular-like myosin cDNA clone may be isolated and its sequence compared to that of the adult ventricular myosin clone.
75
MYOCYTE HYPERTROPHY IN HUM AND RAT HEARTS IS LIMITED WAN ULTIMATE DILUTION OF NUCLEAR DNA TO A VALUE OF 6 pg OF DNA PER 1OOOD p3 OF MYKYTE VOLUME. A. vanderlaarse, H.W. Vliegen, A.V.G. Bruschke. Department of Cardiology, University Hospital, Lelden, The Netherlands. Under normal conditions (growth) as well as under abnormal conditions (pressure or volume overload1 cardiac myocytes have the capacity to enlarge. Rat and human myocytes show ramarkable differences in nuclear adaptation mechanisms in response to hypertrophy. Human myocyte nuclei respond to hypertrophy by polyploidization and multinucleation, thus increasing the DNA content per myocyte from 20 pg (normal) up to 50 pg (severe hypertrophy). In rat hearts with aortic constriction, nuclear DNA content remained constant (13 pgl with reference to normal rat hearts. As myocytes increased in volume, the DNA content per 10000~m3 of qyocyte volume fell,due to dilution, to values never lower than 6 pg/lOOOO ,,m3, in human as well as in rat heart myocytes. We suggest that hypertrophy of myocytes from rat hearts and human hearts is limited by the DNA content per unit of cell volume. Isupported by the Netherlands Heart Foundation)
76
INCREASE IN CALCIUM smmrvm OF AlltIAL SKJMED FIERES FRCN PATIFNTS w?.m DILATH) CARDIUWCPATHY. A. Vankerl, M. F!&IE, I. &xar& , J.C. Riieqg’ i E. Erdmann. Wizinische Klinik I, Klinibm GroShadern, FRG. ‘II.Physiologisches Institut, tiiversit;it Heidelberg, IRG
Fhmirh,
Heart failme muses reduwd effects of B-adremxeptor agonists. %w+over, the calciwrrinduced positive inotropic respmse is slightly reduced. We have investigated whether there is also d change in calcium smsitivity Ka-s) of myofilaments in hearts fran patients with heart failure. Right atria1 ,ml left ventricular fibres of 22 patients with coronary heart disease (CHD)! mitral valve rliseaw WE), ischaemic cardimthy (ICM) or dilated cardicmyopathy (EM) were incubated vlth cardioplegic solution and then skinned with Tritcm X-100 for 24 h and stored in 50% glywrol . Skinned f&es were investigakd in ATP salt solution containing (II+!): ATP 5, HEPES 50, potassium acetate 80, !QCL 6, ETA or CaEl’A 5, creatine phosphate 10, N& 5, creatine kinase 350 U/ml, pH 7.0, 2BC. Myosin light chain distribution and phxphorylation state were studied by tw-dimensicml polyacrylmide gel electrophoresis. me calcium-force relationship of skinned ventricwlar mardim was the same in MVTJ (Fc,o for CC : 5.79fo.02 $a-wits, 101, I01 ( 5.76to.04, 4) and TKM ( 5.789.02, 8). In mmtrast, atria1 fibres of mtients WE 8) than thme of CHD (5.64tO.01, 5), MVll significantly (~(0.05) mxe sensitive for Ca*+ (Echo: 5.7BO.02, (5.65Sl.02, 5) CB ICN (5.63+0.01, 4). 7%us, the differene in Ca-s of atrim and ventricle (abIt 0.11 $aunits) is markedly diminished in KM (0.06). The increased Ca-s of atria1 fibr2s fro0 Kf+patims can nrst b+ due to an increased ptmphotylation of mywin P-light chains &il.CZ) because twc-dimensicml gel electrophoresis pointed out a cmplete dephospbmylation in all skinned fihres preparations. Furthermore, ventriclespecific form of W2 mild be detected in atrial tissue of all gmups in dependence on sw.rity of h+art failure, wtrereas MI atrium-specific MU2 was demnstrahle in any ventricular sample.
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75
MYOCYTE HYPERTROPHY IN HUM AND RAT HEARTS IS LIMITED WAN ULTIMATE DILUTION OF NUCLEAR DNA TO A VALUE OF 6 pg OF DNA PER 1OOOD p3 OF MYKYTE VOLUME. A. vanderlaarse, H.W. Vliegen, A.V.G. Bruschke. Department of Cardiology, University Hospital, Lelden, The Netherlands. Under normal conditions (growth) as well as under abnormal conditions (pressure or volume overload1 cardiac myocytes have the capacity to enlarge. Rat and human myocytes show ramarkable differences in nuclear adaptation mechanisms in response to hypertrophy. Human myocyte nuclei respond to hypertrophy by polyploidization and multinucleation, thus increasing the DNA content per myocyte from 20 pg (normal) up to 50 pg (severe hypertrophy). In rat hearts with aortic constriction, nuclear DNA content remained constant (13 pgl with reference to normal rat hearts. As myocytes increased in volume, the DNA content per 10000~m3 of qyocyte volume fell,due to dilution, to values never lower than 6 pg/lOOOO ,,m3, in human as well as in rat heart myocytes. We suggest that hypertrophy of myocytes from rat hearts and human hearts is limited by the DNA content per unit of cell volume. Isupported by the Netherlands Heart Foundation)
76
INCREASE IN CALCIUM smmrvm OF AlltIAL SKJMED FIERES FRCN PATIFNTS w?.m DILATH) CARDIUWCPATHY. A. Vankerl, M. F!&IE, I. &xar& , J.C. Riieqg’ i E. Erdmann. Wizinische Klinik I, Klinibm GroShadern, FRG. ‘II.Physiologisches Institut, tiiversit;it Heidelberg, IRG
Fhmirh,
Heart failme muses reduwd effects of B-adremxeptor agonists. %w+over, the calciwrrinduced positive inotropic respmse is slightly reduced. We have investigated whether there is also d change in calcium smsitivity Ka-s) of myofilaments in hearts fran patients with heart failure. Right atria1 ,ml left ventricular fibres of 22 patients with coronary heart disease (CHD)! mitral valve rliseaw WE), ischaemic cardimthy (ICM) or dilated cardicmyopathy (EM) were incubated vlth cardioplegic solution and then skinned with Tritcm X-100 for 24 h and stored in 50% glywrol . Skinned f&es were investigakd in ATP salt solution containing (II+!): ATP 5, HEPES 50, potassium acetate 80, !QCL 6, ETA or CaEl’A 5, creatine phosphate 10, N& 5, creatine kinase 350 U/ml, pH 7.0, 2BC. Myosin light chain distribution and phxphorylation state were studied by tw-dimensicml polyacrylmide gel electrophoresis. me calcium-force relationship of skinned ventricwlar mardim was the same in MVTJ (Fc,o for CC : 5.79fo.02 $a-wits, 101, I01 ( 5.76to.04, 4) and TKM ( 5.789.02, 8). In mmtrast, atria1 fibres of mtients WE 8) than thme of CHD (5.64tO.01, 5), MVll significantly (~(0.05) mxe sensitive for Ca*+ (Echo: 5.7BO.02, (5.65Sl.02, 5) CB ICN (5.63+0.01, 4). 7%us, the differene in Ca-s of atrim and ventricle (abIt 0.11 $aunits) is markedly diminished in KM (0.06). The increased Ca-s of atria1 fibr2s fro0 Kf+patims can nrst b+ due to an increased ptmphotylation of mywin P-light chains &il.CZ) because twc-dimensicml gel electrophoresis pointed out a cmplete dephospbmylation in all skinned fihres preparations. Furthermore, ventriclespecific form of W2 mild be detected in atrial tissue of all gmups in dependence on sw.rity of h+art failure, wtrereas MI atrium-specific MU2 was demnstrahle in any ventricular sample.
S.27