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ETOH protection against cytoskeletal damage in cardiomyocytes mediated by NFκB
CONTRACTILE DYSFUNCTION ACCOMPANIED BY ALTERED GENE EXPRESSION AND CA’+-HANDLING IN A RAT MODEL OF DIABETIC CARDIOMYOPATHY Han A.A. van Heugten, Karel Bezstarosti, Ruud van Hof, Regien G. Schoemaker B Jos M. J. Lamers. Inst. Biochemistry 8 Pharmacology, Cardiovasc. Res. Inst. COEUR, Fat. Med. 8 Hlth. Sci., Erasmus University Rotterdam, The Netherlands.
Diabetes mellitus is an independent risk factor for worsening of heart failure. Diabetes-associated changes in cardiac function and RNA/protein expression were studied in the rat model of streptozotocin-induced diabetes meltitus. After 6-8 weeks HbAlc was doubled and blood glucose increased 4-fold, clear signs of diabetes mellitus. No signs of cardiac hypertrophy were detected. Heart function (control rats, n=7, diabetic, n=8) was established in isolated perfused hearts. Basal contractile parameters showed decreased LVSP build up (-38%) while contraction and relaxation rates were decreased as well (+dP/dt -42% and -dP/dt -46%). In contrast to control rats, increased pacing rate did not result in increased LVSP in diabetic rats. Furthermore, the LVEDP-LVSP curve for diabetic rats was significantly attenuated. Biochemical analysis of LV tissue showed decreased ATP-dependent sarcoplasmic reticulum Ca”-uptake, without a change in pCa50. Cardiac gene expression analysis by northern blot showed a 50 % reduction in SERCA2 expression in all heart chambers, while phospholamban (PL) and calsequestrin were not altered. ANF was induced in the ventricles, while BNF was increased in the atria only, Western blot analysis revealed that protein levels of PL were not changed. In summary, these data indicate that one of the underlyinq causes of diabetic contractile dysfunction resides in decreased expression of SERCA2 and increased PUSERCA2 ratio, resulting in decreased SR Ca*‘-uptake and probably reduced SR Ca -storage. Supported by the Dutch Diabetes Foundation, grant nr 98.120
ETOH PROTECTION AGAINST CYTOSKELETAL DAMAGE IN CARDIOMYOCYTES MEDIATED BY NFKB W.B. Van Winkle, M.B. Snuggs, C. Jones and L.M. Buja. Dept. of Pathology, UTx Medical School Houston, TX, USA. Extensive studies support the contention that exposure to low to moderate concentrations of ethanol (ETOH) produces a cytoprotective effect(s) with respect to myocardial ischemia. While a number of possibilities of this ETOH cytoprotective effect, such as changes in coronary vasculature, have been suggested, we have observed ETOH cytoprotection in the myocyte itself. We have found that, following 30-60 min of hypoxia (02 2-40/o), the actin capping cytoskeletal adhesion protein, tensin, is proteolyzed through a complex pathway involving tyrosyl phosphorylation and calpain. We report here that prior exposure of cultured cardiomyocytes to low (5-10 mM) ETOH protects tensin from degradative processes. Concomitantly, ETOH induces the activation of the transcription factor NFKB, as observed both by confocal microscopy and gel shift analysis, Recent studies indicate that this activation pathway involves a pp60csrc-dependent component The possibility that the NFKB cascade is involved in cardiac cytoprotection by low levels of ETOH will be discussed. Supported by NM NIAAA 1228 1.
PREVENTION OF HYPERTROPHY IMPROVES CAPIUAY DENSITY AND ISCHEMIC TOLERANCE IN RAT HEARTS Roeland Van Kerckhoven, Pramod.R. Saxena, Reg1en.G. Schoemaker. Dept of Pharmacology, Erasmus University Rotterdam, The Netherlands Myocardial infarction (MI) induced hypertrophy may decrease capillary density and, hence, ischemic tolerance of the spared myocardium. The present study investigated the effects of moxonldine, a centrally-acting sympatholytic, on myccardial hypertrophy and capillary density in relation to sensitivity to ixhemla. Rats, subjected to coronary artery ligation, were treated with 6 mg/kg/day moxonidine from 1 to 21 days after MI. Then, hearts were isolated, perfused at 85 mmlig and paced at 350 bpm. Left ventricular (LV) dysfunction in MI-hearts, as reflected by a significant decrease in LV systolic pressure (MI: 51*7 vs Sham: 77k6 mmHg), was not improved by moxonidine (44*5 mmHg). Low-flow ischemia was induced bv lowerina cerfuslon oressure to 15 mmHg during 30 min. Sensitivl~ to is&smia, measured as the appearance of ATP catabolites (Sham: 7.6*1.6;MI: 7.5t2.2 uM) and lactate (Sham: 690*122; MI: 595i94 PM) in the coronary effluent, was substantially reduced after moxonldine (Mox: 2.6kO.7 and 29&53 PM, respectively). Ischemic sensitivity, as reflected by maximal coronary flow during reperfusion, was increased in untreated MI-hearts (MI: 19.w.9 vs Sham: 17kO.6 ml/min; P
DOWNREGULATING ACTIVE PYRUVATE DEHYDROGENASE (PDH) COMPLEX ACTIVITY BY OVEREXPRESSING RAT PDH McNitt. Dept. Cellular & Molecular Physiology, Penn State Univ. College of Medicine, Hershey, PA 17033 USA. Pyruvate dehydrogenase kinase 2 (PDK2) is one isoform of a family of kinases that phosphorylate, and inactivate the PDH complex. The effects of overexpression of the PDK isoenzymes per se on PDH complex activity in cardiac muscle have not been reported. The -present studies investigated whether overexpression of PDK2 modulates the regulation of myocardial PDH complex activity in vivo. To accomplish this, the murine cardiac alphamyosin heavy chain (MHC) promoter was used to direct constitutive expression of a rat PDK2 cDNA transgene in B6D2Fl mice. Northern analysis of RNA from the hearts of MHC-PDK2 tra ic mice revealed a robust expression of rat PDK T mRNA. EXDreSSiOn of rat PDK2 did not sionificantlvalter total myocardial PDH activity but redu&d the proportion of active PDH comolex bv 60% comoared with littermate controls. Didhkxoacetate (DCA) ‘inhibits the PDH kinase, and activates PDH complex. The concentration dependency for activation of PDH by DCA showed a decreased sensitivity in tran ic mice. We conclude overexpression of TP K2 reduces active PDH complex and diminishes the sensitivity of the PDH complex to inhibition by DCA. Al79
Diabetes mellitus is an independent risk factor for worsening of heart failure. Diabetes-associated changes in cardiac function and RNA/protein expression were studied in the rat model of streptozotocin-induced diabetes meltitus. After 6-8 weeks HbAlc was doubled and blood glucose increased 4-fold, clear signs of diabetes mellitus. No signs of cardiac hypertrophy were detected. Heart function (control rats, n=7, diabetic, n=8) was established in isolated perfused hearts. Basal contractile parameters showed decreased LVSP build up (-38%) while contraction and relaxation rates were decreased as well (+dP/dt -42% and -dP/dt -46%). In contrast to control rats, increased pacing rate did not result in increased LVSP in diabetic rats. Furthermore, the LVEDP-LVSP curve for diabetic rats was significantly attenuated. Biochemical analysis of LV tissue showed decreased ATP-dependent sarcoplasmic reticulum Ca”-uptake, without a change in pCa50. Cardiac gene expression analysis by northern blot showed a 50 % reduction in SERCA2 expression in all heart chambers, while phospholamban (PL) and calsequestrin were not altered. ANF was induced in the ventricles, while BNF was increased in the atria only, Western blot analysis revealed that protein levels of PL were not changed. In summary, these data indicate that one of the underlyinq causes of diabetic contractile dysfunction resides in decreased expression of SERCA2 and increased PUSERCA2 ratio, resulting in decreased SR Ca*‘-uptake and probably reduced SR Ca -storage. Supported by the Dutch Diabetes Foundation, grant nr 98.120
ETOH PROTECTION AGAINST CYTOSKELETAL DAMAGE IN CARDIOMYOCYTES MEDIATED BY NFKB W.B. Van Winkle, M.B. Snuggs, C. Jones and L.M. Buja. Dept. of Pathology, UTx Medical School Houston, TX, USA. Extensive studies support the contention that exposure to low to moderate concentrations of ethanol (ETOH) produces a cytoprotective effect(s) with respect to myocardial ischemia. While a number of possibilities of this ETOH cytoprotective effect, such as changes in coronary vasculature, have been suggested, we have observed ETOH cytoprotection in the myocyte itself. We have found that, following 30-60 min of hypoxia (02 2-40/o), the actin capping cytoskeletal adhesion protein, tensin, is proteolyzed through a complex pathway involving tyrosyl phosphorylation and calpain. We report here that prior exposure of cultured cardiomyocytes to low (5-10 mM) ETOH protects tensin from degradative processes. Concomitantly, ETOH induces the activation of the transcription factor NFKB, as observed both by confocal microscopy and gel shift analysis, Recent studies indicate that this activation pathway involves a pp60csrc-dependent component The possibility that the NFKB cascade is involved in cardiac cytoprotection by low levels of ETOH will be discussed. Supported by NM NIAAA 1228 1.
PREVENTION OF HYPERTROPHY IMPROVES CAPIUAY DENSITY AND ISCHEMIC TOLERANCE IN RAT HEARTS Roeland Van Kerckhoven, Pramod.R. Saxena, Reg1en.G. Schoemaker. Dept of Pharmacology, Erasmus University Rotterdam, The Netherlands Myocardial infarction (MI) induced hypertrophy may decrease capillary density and, hence, ischemic tolerance of the spared myocardium. The present study investigated the effects of moxonldine, a centrally-acting sympatholytic, on myccardial hypertrophy and capillary density in relation to sensitivity to ixhemla. Rats, subjected to coronary artery ligation, were treated with 6 mg/kg/day moxonidine from 1 to 21 days after MI. Then, hearts were isolated, perfused at 85 mmlig and paced at 350 bpm. Left ventricular (LV) dysfunction in MI-hearts, as reflected by a significant decrease in LV systolic pressure (MI: 51*7 vs Sham: 77k6 mmHg), was not improved by moxonidine (44*5 mmHg). Low-flow ischemia was induced bv lowerina cerfuslon oressure to 15 mmHg during 30 min. Sensitivl~ to is&smia, measured as the appearance of ATP catabolites (Sham: 7.6*1.6;MI: 7.5t2.2 uM) and lactate (Sham: 690*122; MI: 595i94 PM) in the coronary effluent, was substantially reduced after moxonldine (Mox: 2.6kO.7 and 29&53 PM, respectively). Ischemic sensitivity, as reflected by maximal coronary flow during reperfusion, was increased in untreated MI-hearts (MI: 19.w.9 vs Sham: 17kO.6 ml/min; P
DOWNREGULATING ACTIVE PYRUVATE DEHYDROGENASE (PDH) COMPLEX ACTIVITY BY OVEREXPRESSING RAT PDH McNitt. Dept. Cellular & Molecular Physiology, Penn State Univ. College of Medicine, Hershey, PA 17033 USA. Pyruvate dehydrogenase kinase 2 (PDK2) is one isoform of a family of kinases that phosphorylate, and inactivate the PDH complex. The effects of overexpression of the PDK isoenzymes per se on PDH complex activity in cardiac muscle have not been reported. The -present studies investigated whether overexpression of PDK2 modulates the regulation of myocardial PDH complex activity in vivo. To accomplish this, the murine cardiac alphamyosin heavy chain (MHC) promoter was used to direct constitutive expression of a rat PDK2 cDNA transgene in B6D2Fl mice. Northern analysis of RNA from the hearts of MHC-PDK2 tra ic mice revealed a robust expression of rat PDK T mRNA. EXDreSSiOn of rat PDK2 did not sionificantlvalter total myocardial PDH activity but redu&d the proportion of active PDH comolex bv 60% comoared with littermate controls. Didhkxoacetate (DCA) ‘inhibits the PDH kinase, and activates PDH complex. The concentration dependency for activation of PDH by DCA showed a decreased sensitivity in tran ic mice. We conclude overexpression of TP K2 reduces active PDH complex and diminishes the sensitivity of the PDH complex to inhibition by DCA. Al79