- Email: [email protected]
The ku protein complex interacts with YY1, is up-regulated in human heart failure and represses alpha myosin heavy chain gene expression and promoter activity
The 8th Annual Scientific Meeting
•
HFSA
S17
Jay N. Cohn New Investigator Award: Basic Science 001
003
The Ku Protein Complex Interacts with YY1, is Up-Regulated in Human Heart Failure and Represses alpha Myosin Heavy Chain Gene Expression and Promoter Activity Carmen C. Sucharov,1 Steve Helmke,2 Stephen Langer,3 Benjamin Perryman,4 Michael R. Bristow,1 Leslie A. Leinwand3; 1Cardiology, University of Colorado Health Sciences Center, Denver, CO; 2Colorado Center for Innovative Proteomics, University of Colorado Health Sciences Center, Denver, CO; 3MCDBiology, University of Colorado at Boulder, Boulder, CO; 4Cardiovascular Research Institute, University of South Dakota and Sioux Valley Hospitals and Health Systems, Sioux Falls, SD
A MAP4K4-TRF2 Cycle Amplifies Apoptotic Signals Min Xie,1,2 Dou Zhang,1,2 Hidemasa Oh,1,2 Motoaki Sano,1,2 Lloyd H. Michael,5 Mark L. Entman,5 Michael D. Schneider1,2,3,4; 1Department of Medicine, Baylor College of Medicine, Houston, TX; 2Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX; 3Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; 4Molecular Biology and Biophysics, Baylor College of Medicine, Houston, TX; 5DeBakey Heart Center, Baylor College of Medicine, Houston, TX
Heart failure is characterized at the molecular level by changes in gene expression that result in the repression of adult genes such as aMyHC and SERCA2A and the induction of fetal genes such as bMyHC, ANF and skeletal a-actin. The change in MyHC expression is a logical candidate for affecting cardiac contractility since small changes in isoform composition have been shown to affect contractility of cardiac myocytes and the heart. In single cell experiments, cardiac myocytes expressing only 12% of their MyHC as alpha have 52% greater power output than those expressing only beta. The importance of aMyHC expression in the human heart has been recently emphasized by the finding that a mutation in the aMyHC gene can cause either hypertrophic or dilated cardiomyopathies. The aim of our study is to determine the factors responsible for repression of aMyHC expression in the failing human heart. We have recently showed that the transcription factor YY1 is increased in the Failing Human Heart and represses aMyHC promoter activity. In the current study we show that in addition to binding to YY1, the -370/-350 region of the human aMyHC promoter also binds a second protein complex with 6 fold higher activity in human failing hearts. Through DNA affinity column purification followed by mass spectrometry, we have identified this complex as the heterodimer Ku70/Ku80. Mutations in the binding site for Ku70/80 result in up-regulation of the aMyHC promoter. Coexpression of Ku70 or Ku70 and Ku80 together in neonatal rat ventricular myocytes (NRVM) results in repression of aMyHC promoter activity. Western blots of normal and failing human heart extracts show that Ku70 protein levels are increased 6 fold in the failing heart. Immunoprecipitation experiments show that YY1 interacts with Ku70 and Ku80 and that co-expression of YY1 and Ku70 proteins in NRVM results in increased repression of aMyHC promoter activity. Moreover, NRVM infection with adenoviral constructs expressing Ku70 and Ku80 results in a selective repression of endogenous aMyHC mRNA expression and up-regulation of skeletal a-actin mRNA suggesting that the Ku proteins play an important role in the regulation of components of the fetal gene program in cardiac disease.
Telomeres are specialized DNA-protein structures that cap linear chromosome ends, preventing telomere erosion and end-to-end fusion. Maintenance of the cap depends in part on TRF2 (Telomeric Repeat-binding Factor-2). Telomere dysfunction induced by loss of TRF2 causes apoptosis in cardiomyocytes and other cell types, but its means of actuating apoptosis is unknown. The Ste20-like kinase, HPK/GCK-like kinase (HGK, MAP4K4) is involved in the TNFα signaling pathway in tumor cells and couples to JNK specifically through the apoptotic TGFβ-activated kinase 1 (TAK1, MAP3K7). Here we postulate: (1) There is a HGK-TAK1-JNK apoptotic pathway in cardiomyocytes, and (2) apoptosis caused by telomere dysfunction may signal through the HGK-TAK1-JNK apoptotic pathway in cardiomyocytes. In mouse myocardium, each of four biological signals for cardiomyocyte apoptosis up-regulated the activity of this proximal MAP kinase, HGK: biomechanical stress, ischemia/reperfusion injury, and gain-of-function mutations for TNFα or Gq. In cultured rat neonatal cardiomyocytes, analogous results were seen using oxidative stress, ceramide, doxorubicin, or Gq. The HGK-induced apoptosis occurred through TAK1 and JNK. Hence, activation of this little-studied MAP4K, HGK, is a highly generalizable response to cardiac apoptotic stress signals, both in vitro and in vivo. An especially noteworthy activator of HGK was telomere dysfunction from interference of normal TRF2 function. Evidence placing TRF2 functionally upstream of the apoptotic HGK-TAK1-JNK signaling module includes: (1) Dominant-negative TRF2 or knockdown of endogenous TRF2 by antisense oligonucleotides activated HGK, (2) Wild-type TRF2 reduced basal HGK activity, and (3) Apoptosis due to dominant-negative TRF2 was largely blocked by dominant-negative mutations of TAK1 or JNK1. Thus, HGK and MAP kinases downstream of HGK have functional importance in apoptosis caused by telomere dysfunction. Furthermore, the activation of the HGK-TAK1-JNK pathway, in turn, markedly reduced TRF2 levels. Dominant-negative mutations of the pathway protected TRF2 loss induced by ceramide, a potent activator of HGK. Because that loss of TRF2 activated the HGK-TAK1-JNK pathway and activation of the HGKTAK1-JNK pathway induced TRF2 loss, our findings point to the interpretation that telomere dysfunction from loss of TRF2 is reciprocally coupled to activity of the HGK apoptosis pathway. Thus, a positive feedback loop of TRF2 and HGK amplifies the apoptotic signals in cardiomyocytes under stress.
002
004
The Telomere Capping Protein Telomeric Repeat-Binding Factor-2 (TRF2) Mediates Normal Cardiac Muscle Cell Survival and Protects against Doxorubicin-Induced Cardiomyopathy Sam C. Wang,1,2 Arun Prahash,1,2 Hidemasa Oh,3 Motoaki Sano,1,2 Min Xie,1,2 George E. Taffet,2,4 Michael D. Schneider1,2; 1Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX; 2Department of Medicine, Baylor College of Medicine, Houston, TX; 3Department of Experimental Therpeutics, Kyoto University School of Medicine, Kyoto, Japan; 4The DeBakey Heart Center, Baylor College of Medicine, Houston, TX
Natriuretic and Anti-Aldosterone Actions of Chronic Oral Neutral Endopeptidase Inhibition during the Progression of Experimental Congestive Heart Failure Fernando L. Martin,1 Alessandro Cataliotti,1 Tracy L. Stevens,2 John A. Schirger,1 Denise H. Heublein,1 Lisa C. Costello-Boerrigter,1 Guido Boerrigter,1 Margaret M. Redfield,1 John C. Burnett1; 1Cardiorenal Research Laboratory, Mayo Clinic, Rochester, MN; 2Cardiovascular Consultants, Kansas City, MO
Heart failure is a principal source of morbidity and mortality from cardiovascular disease. Apoptosis is one of the causes that reduce functional cardiomyocytes, which may culminate in pump failure. Unfortunately, the cues and mediators that trigger ventricular myocyte death remain poorly understood. Previous work in cultured cardiomyocytes implicates telomere dysfunction, associated with the partial loss of the protein telomeric repeat-binding factor-2 (TRF2), as a cause of apoptosis. Telomeres are composed of repeating TTAGGG tracts with a 3’ single-stranded guanine-rich overhang. The telomere loops back upon itself, allowing the overhang to insert into the upstream telomeric tract to form the T-loop. In this manner, telomeres “cap” the ends of linear chromosomes and shield the leading strand ends from being sensed as double stranded breaks, which would activate the DNA damage response leading to senescence or apoptosis. TRF2 binds to the loop and plays an essential role in preserving the capped state. We investigated the potential role played by TRF2 in myocardium utilizing transgenic mice that over-expressed either wild-type TRF2 (wtTRF2) or a dominant-negative mutant that was created by deleting both the Nterminal basic region and DNA-binding myb motif. The alpha myosin heavy chain promoter was used to express the transgenes in a cardiac-specific manner. Dominantnegative interference of endogenous TRF2 resulted in cardiomyocytes apoptosis, dilated cardiomyopathy with decreased left ventricular function, and early mortality. wtTRF2 mice were challenged with doxorubicin (dox), a chemotherapeutic agent with significant cardiotoxicity, to test the potential therapeutic benefits of TRF2 overexpression. Compared to non-transgenic littermates, heart-specific expression of TRF2 suppressed apoptosis and increased survival after dox treatment. In cultured cardiomyocytes, adenoviral delivery of wild-type TRF2 blunted dox-induced phosphorylation of histone 2AX at serine-15 and p53 at serine-139, target residues for ataxia telangectasia mutated (ATM), the DNA damage sensor. TRF2 also blocked dox-induced apoptosis, as measured by caspase-3 activity and hypodiploidity. These results suggest 1) endogenous TRF2 is important for normal cardiac myocyte viability, and 2) exogenous TRF2 mitigates dox-induced cardiotoxicity, possibly by blunting the activation of the ATMdependent DNA damage pathway.
Background: Neutral endopeptidase (NEP) degrades BNP and ANP which via cGMP are natriuretic, vasodilating, and growth-inhibiting. Studies also show that the natriuretic peptide receptor A which binds both ANP and BNP is highly expressed in the adrenal gland consistent with the aldosterone (ALDO) inhibiting properties of ANP and BNP. Recognizing the early activation of BNP and ANP during progression of congestive heart failure (CHF), we hypothesized chronic oral administration of NEP inhibition (NEPI) initiated in early CHF potentiates the endogenous natriuretic peptide system and delays the onset and magnitude of sodium retention and activation of ALDO during progression to severe CHF. We also hypothesized that chronic NEPI during progressive left ventricular dysfunction (PLVD) improves the natriuretic response to acute volume expansion in severe CHF. Methods: In a canine model which progresses over 38 days from early left ventricular dysfunction to moderate and severe CHF and co-activates ANP and BNP, we defined the actions of chronic NEPI (candoxatril, 10 ng/kg, po beginning day 5) on sodium homeostasis compared to an untreated group (control, CTRL). At the end of the experimental phase, acute volume expansion was induced in all dogs. Results: From baseline to the end of the moderate phase of CHF (day 24), NEPI maintained sodium excretion compared to CTRL (35 ⫾ 3 to 35 ⫾ 5 vs 41 ⫾ 3 to 33 ⫾ 2 mEq/24 hr; p ⬍ 0.05), thus delaying the onset of sodium retention. At this phase, LVEF was higher in the NEPI group compared to CTRL (29 ⫾ 1 vs 22 ⫾ 2, p ⬍ 0.05). In severe CHF sodium excretion was higher in the NEPI group compared to CTRL (21 ⫾ 4 vs 10 ⫾ 4 mEq/24 hr; p ⬍ 0.05) while the natriuretic response to acute volume expansion was enhanced (∆ 933 ⫾ 64 vs 528 ⫾ 210 µEq/min; p ⬍ 0.05). NEPI also resulted in lower plasma ALDO (baseline 3 ⫾ 2 to severe CHF 4 ⫾ 2 vs baseline 1 ⫾ 1 to severe CHF 26 ⫾ 10 pg/dL; p ⬍ 0.05) as compared to CTRL. Conclusion: This study demonstrates that NEPI delays the onset and magnitude of sodium retention in association with a higher level of ventricular systolic function during PLVD. In addition, NEPI suppressed ALDO activation during the progression of CHF and improved the impaired natriuretic response to acute volume expansion in severe CHF. Thus, NEPI emerges as a potential natriuretic and anti-ALDO strategy in CHF that warrants further investigation.
•
HFSA
S17
Jay N. Cohn New Investigator Award: Basic Science 001
003
The Ku Protein Complex Interacts with YY1, is Up-Regulated in Human Heart Failure and Represses alpha Myosin Heavy Chain Gene Expression and Promoter Activity Carmen C. Sucharov,1 Steve Helmke,2 Stephen Langer,3 Benjamin Perryman,4 Michael R. Bristow,1 Leslie A. Leinwand3; 1Cardiology, University of Colorado Health Sciences Center, Denver, CO; 2Colorado Center for Innovative Proteomics, University of Colorado Health Sciences Center, Denver, CO; 3MCDBiology, University of Colorado at Boulder, Boulder, CO; 4Cardiovascular Research Institute, University of South Dakota and Sioux Valley Hospitals and Health Systems, Sioux Falls, SD
A MAP4K4-TRF2 Cycle Amplifies Apoptotic Signals Min Xie,1,2 Dou Zhang,1,2 Hidemasa Oh,1,2 Motoaki Sano,1,2 Lloyd H. Michael,5 Mark L. Entman,5 Michael D. Schneider1,2,3,4; 1Department of Medicine, Baylor College of Medicine, Houston, TX; 2Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX; 3Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; 4Molecular Biology and Biophysics, Baylor College of Medicine, Houston, TX; 5DeBakey Heart Center, Baylor College of Medicine, Houston, TX
Heart failure is characterized at the molecular level by changes in gene expression that result in the repression of adult genes such as aMyHC and SERCA2A and the induction of fetal genes such as bMyHC, ANF and skeletal a-actin. The change in MyHC expression is a logical candidate for affecting cardiac contractility since small changes in isoform composition have been shown to affect contractility of cardiac myocytes and the heart. In single cell experiments, cardiac myocytes expressing only 12% of their MyHC as alpha have 52% greater power output than those expressing only beta. The importance of aMyHC expression in the human heart has been recently emphasized by the finding that a mutation in the aMyHC gene can cause either hypertrophic or dilated cardiomyopathies. The aim of our study is to determine the factors responsible for repression of aMyHC expression in the failing human heart. We have recently showed that the transcription factor YY1 is increased in the Failing Human Heart and represses aMyHC promoter activity. In the current study we show that in addition to binding to YY1, the -370/-350 region of the human aMyHC promoter also binds a second protein complex with 6 fold higher activity in human failing hearts. Through DNA affinity column purification followed by mass spectrometry, we have identified this complex as the heterodimer Ku70/Ku80. Mutations in the binding site for Ku70/80 result in up-regulation of the aMyHC promoter. Coexpression of Ku70 or Ku70 and Ku80 together in neonatal rat ventricular myocytes (NRVM) results in repression of aMyHC promoter activity. Western blots of normal and failing human heart extracts show that Ku70 protein levels are increased 6 fold in the failing heart. Immunoprecipitation experiments show that YY1 interacts with Ku70 and Ku80 and that co-expression of YY1 and Ku70 proteins in NRVM results in increased repression of aMyHC promoter activity. Moreover, NRVM infection with adenoviral constructs expressing Ku70 and Ku80 results in a selective repression of endogenous aMyHC mRNA expression and up-regulation of skeletal a-actin mRNA suggesting that the Ku proteins play an important role in the regulation of components of the fetal gene program in cardiac disease.
Telomeres are specialized DNA-protein structures that cap linear chromosome ends, preventing telomere erosion and end-to-end fusion. Maintenance of the cap depends in part on TRF2 (Telomeric Repeat-binding Factor-2). Telomere dysfunction induced by loss of TRF2 causes apoptosis in cardiomyocytes and other cell types, but its means of actuating apoptosis is unknown. The Ste20-like kinase, HPK/GCK-like kinase (HGK, MAP4K4) is involved in the TNFα signaling pathway in tumor cells and couples to JNK specifically through the apoptotic TGFβ-activated kinase 1 (TAK1, MAP3K7). Here we postulate: (1) There is a HGK-TAK1-JNK apoptotic pathway in cardiomyocytes, and (2) apoptosis caused by telomere dysfunction may signal through the HGK-TAK1-JNK apoptotic pathway in cardiomyocytes. In mouse myocardium, each of four biological signals for cardiomyocyte apoptosis up-regulated the activity of this proximal MAP kinase, HGK: biomechanical stress, ischemia/reperfusion injury, and gain-of-function mutations for TNFα or Gq. In cultured rat neonatal cardiomyocytes, analogous results were seen using oxidative stress, ceramide, doxorubicin, or Gq. The HGK-induced apoptosis occurred through TAK1 and JNK. Hence, activation of this little-studied MAP4K, HGK, is a highly generalizable response to cardiac apoptotic stress signals, both in vitro and in vivo. An especially noteworthy activator of HGK was telomere dysfunction from interference of normal TRF2 function. Evidence placing TRF2 functionally upstream of the apoptotic HGK-TAK1-JNK signaling module includes: (1) Dominant-negative TRF2 or knockdown of endogenous TRF2 by antisense oligonucleotides activated HGK, (2) Wild-type TRF2 reduced basal HGK activity, and (3) Apoptosis due to dominant-negative TRF2 was largely blocked by dominant-negative mutations of TAK1 or JNK1. Thus, HGK and MAP kinases downstream of HGK have functional importance in apoptosis caused by telomere dysfunction. Furthermore, the activation of the HGK-TAK1-JNK pathway, in turn, markedly reduced TRF2 levels. Dominant-negative mutations of the pathway protected TRF2 loss induced by ceramide, a potent activator of HGK. Because that loss of TRF2 activated the HGK-TAK1-JNK pathway and activation of the HGKTAK1-JNK pathway induced TRF2 loss, our findings point to the interpretation that telomere dysfunction from loss of TRF2 is reciprocally coupled to activity of the HGK apoptosis pathway. Thus, a positive feedback loop of TRF2 and HGK amplifies the apoptotic signals in cardiomyocytes under stress.
002
004
The Telomere Capping Protein Telomeric Repeat-Binding Factor-2 (TRF2) Mediates Normal Cardiac Muscle Cell Survival and Protects against Doxorubicin-Induced Cardiomyopathy Sam C. Wang,1,2 Arun Prahash,1,2 Hidemasa Oh,3 Motoaki Sano,1,2 Min Xie,1,2 George E. Taffet,2,4 Michael D. Schneider1,2; 1Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX; 2Department of Medicine, Baylor College of Medicine, Houston, TX; 3Department of Experimental Therpeutics, Kyoto University School of Medicine, Kyoto, Japan; 4The DeBakey Heart Center, Baylor College of Medicine, Houston, TX
Natriuretic and Anti-Aldosterone Actions of Chronic Oral Neutral Endopeptidase Inhibition during the Progression of Experimental Congestive Heart Failure Fernando L. Martin,1 Alessandro Cataliotti,1 Tracy L. Stevens,2 John A. Schirger,1 Denise H. Heublein,1 Lisa C. Costello-Boerrigter,1 Guido Boerrigter,1 Margaret M. Redfield,1 John C. Burnett1; 1Cardiorenal Research Laboratory, Mayo Clinic, Rochester, MN; 2Cardiovascular Consultants, Kansas City, MO
Heart failure is a principal source of morbidity and mortality from cardiovascular disease. Apoptosis is one of the causes that reduce functional cardiomyocytes, which may culminate in pump failure. Unfortunately, the cues and mediators that trigger ventricular myocyte death remain poorly understood. Previous work in cultured cardiomyocytes implicates telomere dysfunction, associated with the partial loss of the protein telomeric repeat-binding factor-2 (TRF2), as a cause of apoptosis. Telomeres are composed of repeating TTAGGG tracts with a 3’ single-stranded guanine-rich overhang. The telomere loops back upon itself, allowing the overhang to insert into the upstream telomeric tract to form the T-loop. In this manner, telomeres “cap” the ends of linear chromosomes and shield the leading strand ends from being sensed as double stranded breaks, which would activate the DNA damage response leading to senescence or apoptosis. TRF2 binds to the loop and plays an essential role in preserving the capped state. We investigated the potential role played by TRF2 in myocardium utilizing transgenic mice that over-expressed either wild-type TRF2 (wtTRF2) or a dominant-negative mutant that was created by deleting both the Nterminal basic region and DNA-binding myb motif. The alpha myosin heavy chain promoter was used to express the transgenes in a cardiac-specific manner. Dominantnegative interference of endogenous TRF2 resulted in cardiomyocytes apoptosis, dilated cardiomyopathy with decreased left ventricular function, and early mortality. wtTRF2 mice were challenged with doxorubicin (dox), a chemotherapeutic agent with significant cardiotoxicity, to test the potential therapeutic benefits of TRF2 overexpression. Compared to non-transgenic littermates, heart-specific expression of TRF2 suppressed apoptosis and increased survival after dox treatment. In cultured cardiomyocytes, adenoviral delivery of wild-type TRF2 blunted dox-induced phosphorylation of histone 2AX at serine-15 and p53 at serine-139, target residues for ataxia telangectasia mutated (ATM), the DNA damage sensor. TRF2 also blocked dox-induced apoptosis, as measured by caspase-3 activity and hypodiploidity. These results suggest 1) endogenous TRF2 is important for normal cardiac myocyte viability, and 2) exogenous TRF2 mitigates dox-induced cardiotoxicity, possibly by blunting the activation of the ATMdependent DNA damage pathway.
Background: Neutral endopeptidase (NEP) degrades BNP and ANP which via cGMP are natriuretic, vasodilating, and growth-inhibiting. Studies also show that the natriuretic peptide receptor A which binds both ANP and BNP is highly expressed in the adrenal gland consistent with the aldosterone (ALDO) inhibiting properties of ANP and BNP. Recognizing the early activation of BNP and ANP during progression of congestive heart failure (CHF), we hypothesized chronic oral administration of NEP inhibition (NEPI) initiated in early CHF potentiates the endogenous natriuretic peptide system and delays the onset and magnitude of sodium retention and activation of ALDO during progression to severe CHF. We also hypothesized that chronic NEPI during progressive left ventricular dysfunction (PLVD) improves the natriuretic response to acute volume expansion in severe CHF. Methods: In a canine model which progresses over 38 days from early left ventricular dysfunction to moderate and severe CHF and co-activates ANP and BNP, we defined the actions of chronic NEPI (candoxatril, 10 ng/kg, po beginning day 5) on sodium homeostasis compared to an untreated group (control, CTRL). At the end of the experimental phase, acute volume expansion was induced in all dogs. Results: From baseline to the end of the moderate phase of CHF (day 24), NEPI maintained sodium excretion compared to CTRL (35 ⫾ 3 to 35 ⫾ 5 vs 41 ⫾ 3 to 33 ⫾ 2 mEq/24 hr; p ⬍ 0.05), thus delaying the onset of sodium retention. At this phase, LVEF was higher in the NEPI group compared to CTRL (29 ⫾ 1 vs 22 ⫾ 2, p ⬍ 0.05). In severe CHF sodium excretion was higher in the NEPI group compared to CTRL (21 ⫾ 4 vs 10 ⫾ 4 mEq/24 hr; p ⬍ 0.05) while the natriuretic response to acute volume expansion was enhanced (∆ 933 ⫾ 64 vs 528 ⫾ 210 µEq/min; p ⬍ 0.05). NEPI also resulted in lower plasma ALDO (baseline 3 ⫾ 2 to severe CHF 4 ⫾ 2 vs baseline 1 ⫾ 1 to severe CHF 26 ⫾ 10 pg/dL; p ⬍ 0.05) as compared to CTRL. Conclusion: This study demonstrates that NEPI delays the onset and magnitude of sodium retention in association with a higher level of ventricular systolic function during PLVD. In addition, NEPI suppressed ALDO activation during the progression of CHF and improved the impaired natriuretic response to acute volume expansion in severe CHF. Thus, NEPI emerges as a potential natriuretic and anti-ALDO strategy in CHF that warrants further investigation.