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A43. Exosome dependent HSP60 secretion in rat cardiac myocytes
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
pathway following cardiac injury leads to myocyte apoptosis and necrosis. But the mechanisms underlying this pathway are not very clear. Studies from our lab indicate that Isoproterenol (ISO) mediated hypertrophy and fibrosis is associated with the induction of CDK1, a kinase normally absent in the adult myocardium. Further this ISO mediated CDK1 induction and cardiac fibrosis were blocked with h1AR specific blocker metoprolol but not with h2AR specific blocker ICI 118,551. These studies suggested that CDK1 may play a role in heart disease. In light of this we further tested the role of ISO in transcriptional regulation of CDK1 in embryonic cardiomyocyte cultures. We used the human CDK1 promoter deletion constructs cloned upstream of a luciferase reporter gene (CDK1p-luc: 6.2 kb, 1.8 kb, 1.1 kb, 700 bp, 245 bp, 100 bp) to assess the promoter activity. ISO (1 AM) induced luciferase activity by 2 fold from all CDK1p-luc constructs and this effect was abolished by metoprolol (10 AM). This suggested that the ISO responsive element may be located within the 100 bp region. A careful inspection of this region revealed two putative NF-Y binding sites but no cAMP response elements were identified. In other cell types NF-Y site has been implicated in Protein Kinase A mediated transcriptional regulation. Further preliminary immuno-blotting studies with NF-Y subunit (AC) specific antibodies indicated that NF-YA and NF-YC levels are downregulated in the adult heart compared to embryonic heart. Future studies will be aimed at site-directed mutagenesis of NF-Y sites within the 100 bp region as well as studying the effects of ISO on the developmental expression of NF-Y subunits. doi:10.1016/j.yjmcc.2006.03.315
A6. An assessment of the role of reactive oxygen species in norepinephrine-induced apoptosis and hypertrophy of h9c2 cardiac myoblasts M.K. Gupta, V. Neelakantan, S. Mishra, R. Tyagi, A. Dinda, S. Maulik, C. Mukhopadhyay, S.K. Goswami. Jawaharlal Nehru University and AIIMS, New Delhi, India Cardiac myocytes upon exposure to increasing doses of norepinephrine (NE), transit from hypertrophic to apoptotic phenotype. Since reactive oxygen species (ROS) generation is attributed to both the phenomenon, we tested whether an elevation in intracellular ROS level causes such transition. H9c2 cardiac myoblasts upon treatment with hypertrophic and apoptotic doses of NE (2 AM and 100 AM respectively) transiently induced intracellular ROS at a comparable level while 200 AM H2O2, another proapoptotic agonist, showed robust and sustained ROS generation. Upon analysis of a number of redoxresponsive transcription factors as the downstream targets of ROS signaling, we observed that NE (2 AM and 100 AM) and H2O2 (200 AM) were ineffective in inducing NF-nappaB while both the agonists upregulated AP-1 and Nrf-2. However, the extents of induction of AP-1 and Nrf-2 were not in direct correlation with the respective ROS levels. Also, AP-1 activities induced by two doses of NE were intrinsically different as at 2 AM, it primarily induced FosB, while at 100 AM it activated Fra-1. Differential
875
induction of FosB and Fra-1 was also reiterated in adult rat myocardium injected with increasing doses of NE. Therefore, NE induces hypertrophy and apoptosis in cardiac myocytes by distinct redox-signaling rather than a general surge of ROS. doi:10.1016/j.yjmcc.2006.03.316
A124. Gene expression profiling of cardiac muscle in response to ischemia in atkins diet conditions C.A. Gregory, T.N. Masters, A.A. Fokin, F. Robicsek. Heineman Medical Research Laboratories, Carolinas Medical Center, Charlotte, NC, USA Background: Cardiac metabolic alterations caused by Atkins Diet (low carbohydrate, high protein and fat) may challenge the myocardial response to ischemia due to different energy consumption pathways. Methods: In the canine model, 8 animals were maintained on Atkins Diet for 6 weeks while 9 animals on normal diet served as a control. Temporary occlusion of the circumflex coronary artery was used for ischemic challenge of the left ventricular myocardium. Samples of endocardium/epicardium were collected and processed for gene expression profiling using the Affymetrix Canine GeneChipR. Results: Using ArrayAssistR 162 probe sets were significantly (P = 0.01) up or down regulated in experimental animals. Significant analysis at P 0.05 of a subset of 240 metabolic genes, revealed 12 upregulated genes and 1 down regulated gene. PathwayAssistR assigned functions to 8 upregulated genes specifically FABP1, APOC-1, APOC-2, PRPS1, CROT,IDI1, PTPN6, and PGS1. Energy requirements of the cardiac muscle response appear to be handled by upregulation of FABP1 which roles include fatty acid uptake, transport, and metabolism. Additional energy may have been provided by activation of LPL by both APOC-1 and 2. LPL is known to hydrolyze triglycerides thus providing free fatty acid to the cell. APOC-1 and 2 also play a role as a ligand/ bridging factor for receptor-mediated lipoprotein uptake. CROT catalyzes the transfer of fatty acyl groups between CoA and carnitine, critical for mitochondrial transport. Conclusions: The predominance of upregulated genes in lipid metabolic pathways accompanying the Atkins Diet would seemingly reduce the ability of the heart to adequately metabolize carbohydrates during ischemic episodes. doi:10.1016/j.yjmcc.2006.03.317
A43. Exosome dependent HSP60 secretion in rat cardiac myocytes Sanjiv Gupta, A.A. Knowlton. Molecular and Cellular Cardiology, University of California, Davis and Sacramento VA Medical Center, CA HSP60 is an important member of the heat shock protein family, found both in mitochondria and the cytosol. Extracel-
876
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
lular HSP60 has been shown to be a potent danger signal for the innate immune system. Here we show that exosomes, small membrane vesicles that are secreted by various cell types, contribute to the release of HSP60 from adult rat cardiac myocytes in both basal and stress-induced (hypoxia 4 h, reoxygenation 2 h, insufficient to release LDH) states. HSP60 release from the rat myocytes was independent of the common secretary pathway, as Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP60 release. To examine whether exosomes contributed to HSP60 release from myocytes, exosomes were isolated from the media in the basal state and after minor stress. HSP 60 was found in exosomes in the basal state, and both the amount of HSP60 and the amount exosomes released were increased after stress. In addition, we found that HSP60 release from the myocytes occurred via a lipid raft-dependent pathway, as treatment with methyl-hcyclodextrin, a raft disrupting drug, somewhat decreased the amount of HSP 60 released. Treatment of myocytes with the exosome inhibitor, dimethyl-amiloride, decreased the amount of HSP60 released to a much greater extent than the lipid raft inhibitor, methyl-h-cyclodextrin. Exosomes accounted for more than 70% of the HSP60 released in the media by myocytes. HSP60 released in the exosomes was ubiquinated. In conclusion, HSP60 is actively released from cardiac myocytes via an exosomal secretory pathway. Supported by HL077281 and the Department of Veterans Affairs. doi:10.1016/j.yjmcc.2006.03.318
A44. Properties of Ca spark voids in normal and failing myocytes revealed by flash photography Sivan V. Meethal, Timothy J. Kamp, Hector H. Valdivia, Robert A. Haworth. University of Wisconsin, 600 Highland Ave, Madison, WI 53792 Excitation-induced Ca gradients were imaged in isolated rat and dog myocytes by a novel method of flash photography of fluo-3 fluorescence. In normal rat myocytes loaded with fluo3AM, Ca sparks were evident 6msec after stimulation emerging from around t-tubules, as judged by co-localization with di-8ANEPPS staining. Voids in the spark pattern coincided not only with gaps in di-8-ANEPPS staining but also with points of vacuolar fluo-3 labeling, visible in unstimulated cells at the zlines, previously identified as lysosomes. In a cell at rest that showed spontaneous Ca spark activity, baseline [Ca] was on average elevated at sites of vacuolar fluo3 labeling, suggesting a higher level of Ca spark activity at these locations. These results suggest that voids in excitation-induced sparks occur at sites of ttubule turnover, but these sites may still support spontaneous Ca sparks. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low. In myocytes from dogs with failure induced by rapid pacing, the reduced Ca transient was associated with greater beat-to-beat spark variance (Ratio of image intensity variance at ‘‘Spark’’ locations/‘‘No Spark’’ locations: 2.42 T 0.45 and 6.05 T 1.08, mean T SD, for control and heart failure respectively, n = 3 cells/group, P < 0.01). A non-uniform spatial
distribution of Ca sparks was also evident in failing cells, which resulted in Ca gradients at longer times after stimulation. Ca gradients rapidly resolved when t-tubular voids were small, as in normal cells, but persisted when the voids were larger, such as occurred in the failing cells, along with t-tubule depletion and derangement. In heart failure this may cause heterogeneous contraction and contribute to contractile failure. doi:10.1016/j.yjmcc.2006.03.319
A72. Mitochondrial responses of neonatal rat ventricular myocytes exposed to lps Chad Jones, L. Maximilian Buja, Diane L.M. Hickson-Bick. University of Texas Medical School at Houston, TX 77030 Unlike in the isolated adult ventricular cardiomyocyte, exposure of neonatal cells to lipopolysaccharide (LPS) does not induce apoptosis. Neonatal cells exhibit a protected phenotype towards this bacterial toxin. LPS does induce significant changes to the mitochondria of neonatal cells. Upon addition of LPS to culture media, neonatal cells rapidly lose energized mitochondria as observed with the potential sensitive dye TMRE. This generalized loss of mitochondrial membrane potential also exhibits an inverse relationship to a redistribution of uncoupling protein UCP3 from a perinuclear concentration into peripheral mitochondria. No changes were observed in the distribution of UCP2. ATP levels also decrease in these cells. The observed changes in mitochondrial membrane potential, UCP3 distribution and ATP levels are reversed over a period of several hours. The cellular recovery of energized mitochondria is the result of mitochondrial biogenesis. Transcription and replication of mitochondrial DNA (mtDNA) requires TFAM, a nuclear encoded enhancer protein, which in turn can be regulated by transcription factors nuclear respiratory factor 1 (NRF-1) and NRF-2. NRF-1 and NRF-2 also act as transcription factors for nuclear genes that encode respiratory complex subunits. PGC-1a, a transcriptional co-activator, can also interact with NRF-1 to induce mitochondrial biogeneseis. In neonatal rat cardiomyocytes exposed to LPS for several hours, we have observed a time-dependent increase in PGC-1a protein, a nuclear translocation of NRF-1 and an up-regulation of TFAM mRNA. These all indicate that the neonatal cells_ ability to recover its energy capacity requires a co-ordinated expression of both nuclear and mitochondrial genes. Supported by grants DAMD17-01-2-0047 and DAMDW81XWH-04-02-0035. doi:10.1016/j.yjmcc.2006.03.320
A21. Cardiac excitation-contraction coupling is altered in ventricular myocytes from aged male but not female mice S.A. Grandy, S.E. Howlett. Department of Pharmacology, Dalhousie University, Halifax, NS, Canada B3H 1X5 This study characterized age-related alterations in excitationcontraction (EC)-coupling in ventricular myocytes and investi-
pathway following cardiac injury leads to myocyte apoptosis and necrosis. But the mechanisms underlying this pathway are not very clear. Studies from our lab indicate that Isoproterenol (ISO) mediated hypertrophy and fibrosis is associated with the induction of CDK1, a kinase normally absent in the adult myocardium. Further this ISO mediated CDK1 induction and cardiac fibrosis were blocked with h1AR specific blocker metoprolol but not with h2AR specific blocker ICI 118,551. These studies suggested that CDK1 may play a role in heart disease. In light of this we further tested the role of ISO in transcriptional regulation of CDK1 in embryonic cardiomyocyte cultures. We used the human CDK1 promoter deletion constructs cloned upstream of a luciferase reporter gene (CDK1p-luc: 6.2 kb, 1.8 kb, 1.1 kb, 700 bp, 245 bp, 100 bp) to assess the promoter activity. ISO (1 AM) induced luciferase activity by 2 fold from all CDK1p-luc constructs and this effect was abolished by metoprolol (10 AM). This suggested that the ISO responsive element may be located within the 100 bp region. A careful inspection of this region revealed two putative NF-Y binding sites but no cAMP response elements were identified. In other cell types NF-Y site has been implicated in Protein Kinase A mediated transcriptional regulation. Further preliminary immuno-blotting studies with NF-Y subunit (AC) specific antibodies indicated that NF-YA and NF-YC levels are downregulated in the adult heart compared to embryonic heart. Future studies will be aimed at site-directed mutagenesis of NF-Y sites within the 100 bp region as well as studying the effects of ISO on the developmental expression of NF-Y subunits. doi:10.1016/j.yjmcc.2006.03.315
A6. An assessment of the role of reactive oxygen species in norepinephrine-induced apoptosis and hypertrophy of h9c2 cardiac myoblasts M.K. Gupta, V. Neelakantan, S. Mishra, R. Tyagi, A. Dinda, S. Maulik, C. Mukhopadhyay, S.K. Goswami. Jawaharlal Nehru University and AIIMS, New Delhi, India Cardiac myocytes upon exposure to increasing doses of norepinephrine (NE), transit from hypertrophic to apoptotic phenotype. Since reactive oxygen species (ROS) generation is attributed to both the phenomenon, we tested whether an elevation in intracellular ROS level causes such transition. H9c2 cardiac myoblasts upon treatment with hypertrophic and apoptotic doses of NE (2 AM and 100 AM respectively) transiently induced intracellular ROS at a comparable level while 200 AM H2O2, another proapoptotic agonist, showed robust and sustained ROS generation. Upon analysis of a number of redoxresponsive transcription factors as the downstream targets of ROS signaling, we observed that NE (2 AM and 100 AM) and H2O2 (200 AM) were ineffective in inducing NF-nappaB while both the agonists upregulated AP-1 and Nrf-2. However, the extents of induction of AP-1 and Nrf-2 were not in direct correlation with the respective ROS levels. Also, AP-1 activities induced by two doses of NE were intrinsically different as at 2 AM, it primarily induced FosB, while at 100 AM it activated Fra-1. Differential
875
induction of FosB and Fra-1 was also reiterated in adult rat myocardium injected with increasing doses of NE. Therefore, NE induces hypertrophy and apoptosis in cardiac myocytes by distinct redox-signaling rather than a general surge of ROS. doi:10.1016/j.yjmcc.2006.03.316
A124. Gene expression profiling of cardiac muscle in response to ischemia in atkins diet conditions C.A. Gregory, T.N. Masters, A.A. Fokin, F. Robicsek. Heineman Medical Research Laboratories, Carolinas Medical Center, Charlotte, NC, USA Background: Cardiac metabolic alterations caused by Atkins Diet (low carbohydrate, high protein and fat) may challenge the myocardial response to ischemia due to different energy consumption pathways. Methods: In the canine model, 8 animals were maintained on Atkins Diet for 6 weeks while 9 animals on normal diet served as a control. Temporary occlusion of the circumflex coronary artery was used for ischemic challenge of the left ventricular myocardium. Samples of endocardium/epicardium were collected and processed for gene expression profiling using the Affymetrix Canine GeneChipR. Results: Using ArrayAssistR 162 probe sets were significantly (P = 0.01) up or down regulated in experimental animals. Significant analysis at P 0.05 of a subset of 240 metabolic genes, revealed 12 upregulated genes and 1 down regulated gene. PathwayAssistR assigned functions to 8 upregulated genes specifically FABP1, APOC-1, APOC-2, PRPS1, CROT,IDI1, PTPN6, and PGS1. Energy requirements of the cardiac muscle response appear to be handled by upregulation of FABP1 which roles include fatty acid uptake, transport, and metabolism. Additional energy may have been provided by activation of LPL by both APOC-1 and 2. LPL is known to hydrolyze triglycerides thus providing free fatty acid to the cell. APOC-1 and 2 also play a role as a ligand/ bridging factor for receptor-mediated lipoprotein uptake. CROT catalyzes the transfer of fatty acyl groups between CoA and carnitine, critical for mitochondrial transport. Conclusions: The predominance of upregulated genes in lipid metabolic pathways accompanying the Atkins Diet would seemingly reduce the ability of the heart to adequately metabolize carbohydrates during ischemic episodes. doi:10.1016/j.yjmcc.2006.03.317
A43. Exosome dependent HSP60 secretion in rat cardiac myocytes Sanjiv Gupta, A.A. Knowlton. Molecular and Cellular Cardiology, University of California, Davis and Sacramento VA Medical Center, CA HSP60 is an important member of the heat shock protein family, found both in mitochondria and the cytosol. Extracel-
876
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918
lular HSP60 has been shown to be a potent danger signal for the innate immune system. Here we show that exosomes, small membrane vesicles that are secreted by various cell types, contribute to the release of HSP60 from adult rat cardiac myocytes in both basal and stress-induced (hypoxia 4 h, reoxygenation 2 h, insufficient to release LDH) states. HSP60 release from the rat myocytes was independent of the common secretary pathway, as Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP60 release. To examine whether exosomes contributed to HSP60 release from myocytes, exosomes were isolated from the media in the basal state and after minor stress. HSP 60 was found in exosomes in the basal state, and both the amount of HSP60 and the amount exosomes released were increased after stress. In addition, we found that HSP60 release from the myocytes occurred via a lipid raft-dependent pathway, as treatment with methyl-hcyclodextrin, a raft disrupting drug, somewhat decreased the amount of HSP 60 released. Treatment of myocytes with the exosome inhibitor, dimethyl-amiloride, decreased the amount of HSP60 released to a much greater extent than the lipid raft inhibitor, methyl-h-cyclodextrin. Exosomes accounted for more than 70% of the HSP60 released in the media by myocytes. HSP60 released in the exosomes was ubiquinated. In conclusion, HSP60 is actively released from cardiac myocytes via an exosomal secretory pathway. Supported by HL077281 and the Department of Veterans Affairs. doi:10.1016/j.yjmcc.2006.03.318
A44. Properties of Ca spark voids in normal and failing myocytes revealed by flash photography Sivan V. Meethal, Timothy J. Kamp, Hector H. Valdivia, Robert A. Haworth. University of Wisconsin, 600 Highland Ave, Madison, WI 53792 Excitation-induced Ca gradients were imaged in isolated rat and dog myocytes by a novel method of flash photography of fluo-3 fluorescence. In normal rat myocytes loaded with fluo3AM, Ca sparks were evident 6msec after stimulation emerging from around t-tubules, as judged by co-localization with di-8ANEPPS staining. Voids in the spark pattern coincided not only with gaps in di-8-ANEPPS staining but also with points of vacuolar fluo-3 labeling, visible in unstimulated cells at the zlines, previously identified as lysosomes. In a cell at rest that showed spontaneous Ca spark activity, baseline [Ca] was on average elevated at sites of vacuolar fluo3 labeling, suggesting a higher level of Ca spark activity at these locations. These results suggest that voids in excitation-induced sparks occur at sites of ttubule turnover, but these sites may still support spontaneous Ca sparks. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low. In myocytes from dogs with failure induced by rapid pacing, the reduced Ca transient was associated with greater beat-to-beat spark variance (Ratio of image intensity variance at ‘‘Spark’’ locations/‘‘No Spark’’ locations: 2.42 T 0.45 and 6.05 T 1.08, mean T SD, for control and heart failure respectively, n = 3 cells/group, P < 0.01). A non-uniform spatial
distribution of Ca sparks was also evident in failing cells, which resulted in Ca gradients at longer times after stimulation. Ca gradients rapidly resolved when t-tubular voids were small, as in normal cells, but persisted when the voids were larger, such as occurred in the failing cells, along with t-tubule depletion and derangement. In heart failure this may cause heterogeneous contraction and contribute to contractile failure. doi:10.1016/j.yjmcc.2006.03.319
A72. Mitochondrial responses of neonatal rat ventricular myocytes exposed to lps Chad Jones, L. Maximilian Buja, Diane L.M. Hickson-Bick. University of Texas Medical School at Houston, TX 77030 Unlike in the isolated adult ventricular cardiomyocyte, exposure of neonatal cells to lipopolysaccharide (LPS) does not induce apoptosis. Neonatal cells exhibit a protected phenotype towards this bacterial toxin. LPS does induce significant changes to the mitochondria of neonatal cells. Upon addition of LPS to culture media, neonatal cells rapidly lose energized mitochondria as observed with the potential sensitive dye TMRE. This generalized loss of mitochondrial membrane potential also exhibits an inverse relationship to a redistribution of uncoupling protein UCP3 from a perinuclear concentration into peripheral mitochondria. No changes were observed in the distribution of UCP2. ATP levels also decrease in these cells. The observed changes in mitochondrial membrane potential, UCP3 distribution and ATP levels are reversed over a period of several hours. The cellular recovery of energized mitochondria is the result of mitochondrial biogenesis. Transcription and replication of mitochondrial DNA (mtDNA) requires TFAM, a nuclear encoded enhancer protein, which in turn can be regulated by transcription factors nuclear respiratory factor 1 (NRF-1) and NRF-2. NRF-1 and NRF-2 also act as transcription factors for nuclear genes that encode respiratory complex subunits. PGC-1a, a transcriptional co-activator, can also interact with NRF-1 to induce mitochondrial biogeneseis. In neonatal rat cardiomyocytes exposed to LPS for several hours, we have observed a time-dependent increase in PGC-1a protein, a nuclear translocation of NRF-1 and an up-regulation of TFAM mRNA. These all indicate that the neonatal cells_ ability to recover its energy capacity requires a co-ordinated expression of both nuclear and mitochondrial genes. Supported by grants DAMD17-01-2-0047 and DAMDW81XWH-04-02-0035. doi:10.1016/j.yjmcc.2006.03.320
A21. Cardiac excitation-contraction coupling is altered in ventricular myocytes from aged male but not female mice S.A. Grandy, S.E. Howlett. Department of Pharmacology, Dalhousie University, Halifax, NS, Canada B3H 1X5 This study characterized age-related alterations in excitationcontraction (EC)-coupling in ventricular myocytes and investi-