- Email: [email protected]
Expression of Ca2+-handling proteins in aged rat heart
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825
Aim: To determine the development of LV function over an 8 week period following myocardial infarction in mice. To establish the possible induction of D3 activity and to correlate this with LV function over the course of LV remodeling. Methods: We investigated LV remodeling following myocardial infarction in mice. Functional and morphological parameters were obtained at 1, 4 and 8 weeks following MI. Results: MI resulted in an increased LV end-diastolic diameter and decreased fractional shortening. The time constant of isovolumic relaxation was increased, indicative of reduced expression of the TH-responsive Ca-ATPase (SERCA2a). LV end-diastolic pressure was increased at 1 and 4 weeks, but was again normal at 8 weeks. Pulmonary congestion was indicated at 4 weeks, but this resolved at 8 weeks. MI had no effect on heart rate. Serum T3 levels were unaltered, but T4 level was below control at 1 week. D3 activity was determined in the noninfarcted, remodeling LV. MI resulted in a stable, 14–22 fold induction of D3 activity after 1 week. Conclusion: Post-MI remodeling of the LV in mice is associated with a substantial and stable induction of D3 activity. Heart function is maintained in these animals and rather than a factor contributing to failure, the induction of D3 and a tissuespecific hypothyroid condition may be part of an adaptive response. Keywords: Myocardial infarction; Thyroid hormone; Deiodinase type III doi:10.1016/j.yjmcc.2008.02.029
Abstract No. 29 Accumulation of 4-hydroxynonenal protein adducts and Bax protein in rat hearts during aging Peter Kaplan⁎, Zuzana Tatarkova, Monika Sivonova, Jan Lehotsky, Dusan Dobrota. Department of Biochemistry, Jessenius Faculty of Medicine, Martin, Slovakia ⁎ Corresponding author. Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, SK 036 01 Martin, Slovakia. Tel.: +421 043 4131565; fax: +421 043 4136770. E-mail address: [email protected] The accumulation of oxidatively damaged molecules has been implicated to be a major cause of cellular dysfunction that occurs during the aging process. 4-hydroxynonenal (HNE), a major lipid peroxidation (LPO) product, exerts various cytotoxic and signalling effects. The aim of this work was to investigate formation of HNE and HNE-protein adducts in hearts of adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. HNE concentration was markedly elevated in old (134.6 ± 4.5% vs. adult group, p b 0.01) and senescent group (173.5 ± 1.2%, p b 0.001). To assess potential modification of proteins by HNE we performed Western blot analysis with an antibody recognizing HNE-protein Michael adducts. The results showed enhanced
723
formation of HNE adducts with few proteins of heart homogenates from old and senescent rats. The formation of protein adducts with LPO end-products was also confirmed by spectrofluorimetric methods. In 15- and 26-month old rats the fluorescence intensity of lysine residues modified by LPO endproducts increased by 29.0 ± 3.3% ( p b 0.001) and 79.2 ± 8.3% ( p b 0.001), respectively, as compared to 6-month old animals. There is increasing evidence that apoptosis plays an important role in myocardial aging. Because LPO products can be apoptotisinducing signals we also evaluated protein levels of pro-apoptotic regulator Bax. Cardiac Bax levels were significantly higher in 15and 26-month old rats when compared to 6-month old animals. These results suggest that HNE is involved in oxidative modification of proteins during aging; however, further studies are required to determine its role in apoptosis during myocardial aging. This work was supported by grants VEGA SR 1/0027/08 and APVV 51-027404. Keywords: Aging; Oxidative stress; Protein modification doi:10.1016/j.yjmcc.2008.02.030
Abstract No. 30 Expression of Ca2+-handling proteins in aged rat heart Dusan Dobrota⁎, Peter Kaplan, Dana Jurkovicova, Eva Babusikova, Sona Hudecova, Peter Racay, Marta Sirova, Jan Lehotsky, Anna Drgova, Olga Krizanova. Jessenius Faculty of Medicine, Martin, Institute of Molecular Physiology and Genetics, SAS, Bratislava, Slovakia ⁎ Corresponding author. Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, SK 036 01 Martin, Slovakia. Tel.: +421 043 4131565; fax: +421 043 4136770. E-mail address: [email protected] Aging is associated with detrimental structural and functional changes, including disturbance of intracellular Ca2+ homeostasis. In cardiac tissue, the sarcoplasmic reticulum (SR) plays a key role in intracellular Ca2+ regulation and in excitation–contraction coupling. The purpose of the present study was to examine the effect of aging on gene expression and protein levels of SR Ca2+ transport/regulatory proteins — Ca2+-ATPase (SERCA2), ryanodine receptor (RYR), inositol 1,4,5-triphosphate receptor (IP3R), and phospholamban (PLA). The SERCA2 activity decreased progressively during aging, the maximum velocity was reduced by 20% ( p b 0.001) and 27% ( p b 0.001) in hearts of 15-month and 26-month old rats, respectively, compared with 6-month old rats. However, mRNA (determined by RT-PCR) and protein levels (determined by Western blot analysis) of SERCA were not altered during aging. Similarly, there were no significant differences among investigated age-groups in the expression of SERCA2 regulatory protein PLA and Ca2+ channel, type 2 RYR. In contrast, aging was associated with progressive increase
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825
of mRNA levels of type 1 IP3R — Ca2+ release channel. The changes were observed in both atria and ventricles and correlated well with contents of the IP3R1 protein. These results suggest that altered gene expression as well as posttranslational modifications of some SR Ca2+ handling proteins might affect intracellular calcium homeostasis and cardiac function during the aging process. This work was supported by grants VEGA SR 1/0027/08 and APVV 51-027404. Keywords: Aging; Sarcoplasmic reticulum; Gene expression doi:10.1016/j.yjmcc.2008.02.031
Abstract No. 31 Different modulation of type 1 and 2 IP3 receptors by stress and catecholamines Olga Krizanovaa,⁎, Silvia Pastorekovab, Richard Kvetnanskyc. a Institute of Molecular Physiology and Genetics, SAS, Vlarska 5, 833 34 Bratislava, Slovakia. bInstitute of Virology, SAS, Bratislava, Slovakia. cInstitute of Experimental Endocrinology, SAS, Bratislava, Slovakia ⁎ Corresponding author. Tel.: +421 254772211; fax: +421 254773666. E-mail address: [email protected] Inositol 1,4.5-triphosphate receptors are calcium release channels located in the endoplasmic/sarcoplasmic membrane of the cell. In the heart, both type 1 and 2 receptors are present, but differently localized. The type 1 IP3 receptors are most abundant in cardiac ganglia and type 2 IP3 receptors are prevalent in cardiomyocytes. In the heart, gene expression of type 1 IP3 receptor is modulated by catecholamines, while type 2 receptor is not affected. In this study, we compared the effect of two stressors – cold exposure and hypoxia – on the gene expression and protein levels of the type 1 and 2 IP3 receptors. We have shown that both types of IP3 receptors were increased by these stressors, although the response of the type 2 IP3 receptors was delayed. We propose that physiological importance of these types of IP3 receptors in the heart might differ in both, control conditions and during stress. This work was supported by following grants: APVT 51027404 and VEGA 2/6078. Keywords: IP3 receptors; Stress; Catecholamines doi:10.1016/j.yjmcc.2008.02.032
Portnichenko, Olexiy O. Moybenko. Bogomoletz Institute of Physiology, NAS of Ukraine, Kyiv, Ukraine. ICAMER, NAS of Ukraine, Kyiv, Ukraine ⁎ Corresponding author. 4 Bogomoletz Street, 01024 Kyiv, Ukraine. Tel.: +38 44 2562061; fax: +38 44 2562494. E-mail address: [email protected]
Earlier we established the ability of acute systemic (whole body) hypoxia to induce the phenomenon of delayed cardioprotection in rats (Portnychenko et al., 2002). This late hypoxic preconditioning led to infarct size, LDH release, and myocardial apoptosis reduction, as well as strongly prevented fatal arrhythmia incidence in ischemic and reperfused isolated hearts. Transcription factors involved were studied in adult male Wistar rats exposed to the hypoxic preconditioning (normobaric hypoxia with 10% O2 in air, 3 h, or hypobaric hypoxia in barochamber, 5600 m, 3 h). Expression of HIF-1α and HIF-3α mRNA was estimated by RTPCR, and expression of HIF-1α protein — by Western blotting. It was shown that initial HIF-3α mRNA expression was 9.0-fold higher than HIF-1α expression in both ventricles. In the right ventricles expression values were 4.0–8.0-fold higher, than in the left ones. In 24 h after the preconditioning, HIF-1α and HIF-3α mRNA expression strongly elevated (2.4–2.8-fold) in the left ventricles without HIF protein stabilization. The right ventricles demonstrated a significant increase only in HIF-3α mRNA expression (1.4-fold). Thus, the right ventricles have initial higher oxygen sensitivity and gene transcription ability (“native preconditioning”), and less reaction to hypoxical stimuli that is accompanied by more significantly delayed cardioprotection from infarction than in the left ventricles. HIF-3α seems to be more strongly involved in hypoxic preconditioning than HIF-1α. Keywords: Late hypoxic preconditioning; HIF-3α; Left and right ventricles doi:10.1016/j.yjmcc.2008.02.033
Abstract No. 33 Isolated rat ventricular myocytes retain protection afforded by in vivo hypoxic preconditioning or chronic hypoxia Gudrun H. Borchert⁎, Jan Kopecky, Frantisek Kolar. Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. Centre for Cardiovascular Research, Prague, Czech Republic ⁎ Corresponding author. Tel.: +420 241062693; fax: +420 241062125. E-mail address: [email protected]
Abstract No. 32 Expression of HIF-1α and HIF-3α differentially changed in rat heart ventricles after hypoxic preconditioning Alla G. Portnychenko⁎, Viktor E. Dosenko, Volodymyr I.
Both hypoxic preconditioning (HP) and chronic hypoxia (CH) protect the heart against injury caused by acute oxygen deprivation. The aim of the study was to find out whether the protection was preserved in ventricular myocytes isolated after
Aim: To determine the development of LV function over an 8 week period following myocardial infarction in mice. To establish the possible induction of D3 activity and to correlate this with LV function over the course of LV remodeling. Methods: We investigated LV remodeling following myocardial infarction in mice. Functional and morphological parameters were obtained at 1, 4 and 8 weeks following MI. Results: MI resulted in an increased LV end-diastolic diameter and decreased fractional shortening. The time constant of isovolumic relaxation was increased, indicative of reduced expression of the TH-responsive Ca-ATPase (SERCA2a). LV end-diastolic pressure was increased at 1 and 4 weeks, but was again normal at 8 weeks. Pulmonary congestion was indicated at 4 weeks, but this resolved at 8 weeks. MI had no effect on heart rate. Serum T3 levels were unaltered, but T4 level was below control at 1 week. D3 activity was determined in the noninfarcted, remodeling LV. MI resulted in a stable, 14–22 fold induction of D3 activity after 1 week. Conclusion: Post-MI remodeling of the LV in mice is associated with a substantial and stable induction of D3 activity. Heart function is maintained in these animals and rather than a factor contributing to failure, the induction of D3 and a tissuespecific hypothyroid condition may be part of an adaptive response. Keywords: Myocardial infarction; Thyroid hormone; Deiodinase type III doi:10.1016/j.yjmcc.2008.02.029
Abstract No. 29 Accumulation of 4-hydroxynonenal protein adducts and Bax protein in rat hearts during aging Peter Kaplan⁎, Zuzana Tatarkova, Monika Sivonova, Jan Lehotsky, Dusan Dobrota. Department of Biochemistry, Jessenius Faculty of Medicine, Martin, Slovakia ⁎ Corresponding author. Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, SK 036 01 Martin, Slovakia. Tel.: +421 043 4131565; fax: +421 043 4136770. E-mail address: [email protected] The accumulation of oxidatively damaged molecules has been implicated to be a major cause of cellular dysfunction that occurs during the aging process. 4-hydroxynonenal (HNE), a major lipid peroxidation (LPO) product, exerts various cytotoxic and signalling effects. The aim of this work was to investigate formation of HNE and HNE-protein adducts in hearts of adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. HNE concentration was markedly elevated in old (134.6 ± 4.5% vs. adult group, p b 0.01) and senescent group (173.5 ± 1.2%, p b 0.001). To assess potential modification of proteins by HNE we performed Western blot analysis with an antibody recognizing HNE-protein Michael adducts. The results showed enhanced
723
formation of HNE adducts with few proteins of heart homogenates from old and senescent rats. The formation of protein adducts with LPO end-products was also confirmed by spectrofluorimetric methods. In 15- and 26-month old rats the fluorescence intensity of lysine residues modified by LPO endproducts increased by 29.0 ± 3.3% ( p b 0.001) and 79.2 ± 8.3% ( p b 0.001), respectively, as compared to 6-month old animals. There is increasing evidence that apoptosis plays an important role in myocardial aging. Because LPO products can be apoptotisinducing signals we also evaluated protein levels of pro-apoptotic regulator Bax. Cardiac Bax levels were significantly higher in 15and 26-month old rats when compared to 6-month old animals. These results suggest that HNE is involved in oxidative modification of proteins during aging; however, further studies are required to determine its role in apoptosis during myocardial aging. This work was supported by grants VEGA SR 1/0027/08 and APVV 51-027404. Keywords: Aging; Oxidative stress; Protein modification doi:10.1016/j.yjmcc.2008.02.030
Abstract No. 30 Expression of Ca2+-handling proteins in aged rat heart Dusan Dobrota⁎, Peter Kaplan, Dana Jurkovicova, Eva Babusikova, Sona Hudecova, Peter Racay, Marta Sirova, Jan Lehotsky, Anna Drgova, Olga Krizanova. Jessenius Faculty of Medicine, Martin, Institute of Molecular Physiology and Genetics, SAS, Bratislava, Slovakia ⁎ Corresponding author. Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, SK 036 01 Martin, Slovakia. Tel.: +421 043 4131565; fax: +421 043 4136770. E-mail address: [email protected] Aging is associated with detrimental structural and functional changes, including disturbance of intracellular Ca2+ homeostasis. In cardiac tissue, the sarcoplasmic reticulum (SR) plays a key role in intracellular Ca2+ regulation and in excitation–contraction coupling. The purpose of the present study was to examine the effect of aging on gene expression and protein levels of SR Ca2+ transport/regulatory proteins — Ca2+-ATPase (SERCA2), ryanodine receptor (RYR), inositol 1,4,5-triphosphate receptor (IP3R), and phospholamban (PLA). The SERCA2 activity decreased progressively during aging, the maximum velocity was reduced by 20% ( p b 0.001) and 27% ( p b 0.001) in hearts of 15-month and 26-month old rats, respectively, compared with 6-month old rats. However, mRNA (determined by RT-PCR) and protein levels (determined by Western blot analysis) of SERCA were not altered during aging. Similarly, there were no significant differences among investigated age-groups in the expression of SERCA2 regulatory protein PLA and Ca2+ channel, type 2 RYR. In contrast, aging was associated with progressive increase
724
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825
of mRNA levels of type 1 IP3R — Ca2+ release channel. The changes were observed in both atria and ventricles and correlated well with contents of the IP3R1 protein. These results suggest that altered gene expression as well as posttranslational modifications of some SR Ca2+ handling proteins might affect intracellular calcium homeostasis and cardiac function during the aging process. This work was supported by grants VEGA SR 1/0027/08 and APVV 51-027404. Keywords: Aging; Sarcoplasmic reticulum; Gene expression doi:10.1016/j.yjmcc.2008.02.031
Abstract No. 31 Different modulation of type 1 and 2 IP3 receptors by stress and catecholamines Olga Krizanovaa,⁎, Silvia Pastorekovab, Richard Kvetnanskyc. a Institute of Molecular Physiology and Genetics, SAS, Vlarska 5, 833 34 Bratislava, Slovakia. bInstitute of Virology, SAS, Bratislava, Slovakia. cInstitute of Experimental Endocrinology, SAS, Bratislava, Slovakia ⁎ Corresponding author. Tel.: +421 254772211; fax: +421 254773666. E-mail address: [email protected] Inositol 1,4.5-triphosphate receptors are calcium release channels located in the endoplasmic/sarcoplasmic membrane of the cell. In the heart, both type 1 and 2 receptors are present, but differently localized. The type 1 IP3 receptors are most abundant in cardiac ganglia and type 2 IP3 receptors are prevalent in cardiomyocytes. In the heart, gene expression of type 1 IP3 receptor is modulated by catecholamines, while type 2 receptor is not affected. In this study, we compared the effect of two stressors – cold exposure and hypoxia – on the gene expression and protein levels of the type 1 and 2 IP3 receptors. We have shown that both types of IP3 receptors were increased by these stressors, although the response of the type 2 IP3 receptors was delayed. We propose that physiological importance of these types of IP3 receptors in the heart might differ in both, control conditions and during stress. This work was supported by following grants: APVT 51027404 and VEGA 2/6078. Keywords: IP3 receptors; Stress; Catecholamines doi:10.1016/j.yjmcc.2008.02.032
Portnichenko, Olexiy O. Moybenko. Bogomoletz Institute of Physiology, NAS of Ukraine, Kyiv, Ukraine. ICAMER, NAS of Ukraine, Kyiv, Ukraine ⁎ Corresponding author. 4 Bogomoletz Street, 01024 Kyiv, Ukraine. Tel.: +38 44 2562061; fax: +38 44 2562494. E-mail address: [email protected]
Earlier we established the ability of acute systemic (whole body) hypoxia to induce the phenomenon of delayed cardioprotection in rats (Portnychenko et al., 2002). This late hypoxic preconditioning led to infarct size, LDH release, and myocardial apoptosis reduction, as well as strongly prevented fatal arrhythmia incidence in ischemic and reperfused isolated hearts. Transcription factors involved were studied in adult male Wistar rats exposed to the hypoxic preconditioning (normobaric hypoxia with 10% O2 in air, 3 h, or hypobaric hypoxia in barochamber, 5600 m, 3 h). Expression of HIF-1α and HIF-3α mRNA was estimated by RTPCR, and expression of HIF-1α protein — by Western blotting. It was shown that initial HIF-3α mRNA expression was 9.0-fold higher than HIF-1α expression in both ventricles. In the right ventricles expression values were 4.0–8.0-fold higher, than in the left ones. In 24 h after the preconditioning, HIF-1α and HIF-3α mRNA expression strongly elevated (2.4–2.8-fold) in the left ventricles without HIF protein stabilization. The right ventricles demonstrated a significant increase only in HIF-3α mRNA expression (1.4-fold). Thus, the right ventricles have initial higher oxygen sensitivity and gene transcription ability (“native preconditioning”), and less reaction to hypoxical stimuli that is accompanied by more significantly delayed cardioprotection from infarction than in the left ventricles. HIF-3α seems to be more strongly involved in hypoxic preconditioning than HIF-1α. Keywords: Late hypoxic preconditioning; HIF-3α; Left and right ventricles doi:10.1016/j.yjmcc.2008.02.033
Abstract No. 33 Isolated rat ventricular myocytes retain protection afforded by in vivo hypoxic preconditioning or chronic hypoxia Gudrun H. Borchert⁎, Jan Kopecky, Frantisek Kolar. Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. Centre for Cardiovascular Research, Prague, Czech Republic ⁎ Corresponding author. Tel.: +420 241062693; fax: +420 241062125. E-mail address: [email protected]
Abstract No. 32 Expression of HIF-1α and HIF-3α differentially changed in rat heart ventricles after hypoxic preconditioning Alla G. Portnychenko⁎, Viktor E. Dosenko, Volodymyr I.
Both hypoxic preconditioning (HP) and chronic hypoxia (CH) protect the heart against injury caused by acute oxygen deprivation. The aim of the study was to find out whether the protection was preserved in ventricular myocytes isolated after