- Email: [email protected]
Restoration of contractile function in isolated failing human ventricular cardiac myocytes by gene transfer of SERCA2a
Journal of Cardiac Failure Vol. 5 No. 3 Suppl. 1 1999
•
1
Young Investigator Awards Y1
Y2
Restoration of Contractile Function in Isolated Failing Human Ventricular Cardiac Myocytes by Gene Transfer of SERCA2a Federica del Monte, Sian Harding, Judith K. Gwathmey, Anthony Rosenzweig, Roger J. Hajjar; Cardiov. Res. Center, Mass. Gen. Hosp, Boston Univ., Boston, MA, Imperial College, London
Regulation of Tumor Necrosis Factor by Volume and Pressure Overload in Human Myocardium: Effect of Chronic Ventricular Unloading and Pressure Overload Elimination Guillermo Torre-Amione, Sonny S. Stetson, Keith A. Youker, Jean-Bernard Durand, Sherif F. Nagueh, William H. Spencer, III, Mark L. Entman; Cardiology, Baylor College of Medicine, Houston, TX
Myocardial cells from failing (F) human hearts are characterized by abnormal Ca2+ handling, a negative force frequency relationship (FFR), and decreased sarcoplasmic reticulum (SR) ATPase activity. We have previously shown that overexpmssion of SERCA2a by adenoviral gene transfer increases SR ATPase activity and enhances Ca2+ handling both in vitro and in vivo. To test whether contractile function can be restored by overexpression of SERCA2a in human hearts, myocardial cells isolated from 10 patients with end-stage heart failure were infected with adenoviruses encoding either the SERCA2a gene or reporter genes green fluorescent protein (GFP) and /3-galactosidase for 24 hrs. Cells isolated from 2 non-failing hearts were used as controls (C). As shown below, compared to C myocytes, F myocardial cells had prolonged contraction and calcium transient detected by Fura-2. Overexpression of SERCA2a in F myocytes decreased the time course of both contractions and Caz+ by 19+2% and 27-+4%. F myocytes had decreased Ca2+ release and an increase in diastolic Ca 2+ with increasing frequency (0.1-1.5Hz). Overexpression of SERCA2a restored the FFR in the F cells to normal: increasing frequency resulted in enhanced SR Ca 2+ release and contraction. These results show that gene transfer of SERCA2a improves the contractile function in F human myocytes. Targeting SERCA2a may provide therapeutic benefits in heart failure. Non F a i l i n g
Failing - GFP
Failing - SERCA
An increasing number of observations suggest that TNF plays a pathogenetic role in heart failure; however, the mechanisms that regulate myocardial (m) TNF production in humans are not known. Under experimental conditions, volume and pressure overload stimulate mTNF production; accordingly, the goal of our studies was to determine the effect of changes in ventricular volume and pressure on mTNF expression and its relationship to cardiac function in men. To accomplish this, mTNF concentrations were determined in two distinct patient populations. First, in LV samples from patients with dialated cardiomyopathy(DCM) before and after LVAD implantation, n - 8(volume model). Second, in RV septal biopsies obtained from patients with hypertorphic obstructive cardiomyopathy(HOCM) before and after pressure gradient elimination by ethanol induced septal ablation, n=17(pressure model). TNF content was determined by immunohistochemistry and by semi-quantitative analysis of stained areas. LVAD treated patients had a 42% ± 9% reduction in mTNF concentration after chronic ventricular unloading with LVADs, whereas in HOCM patients, mTNF concentration was reduced to 49.5%-+14% after pressure gradient elimination. We found decreased mTNF content in all 25 paired myocardial samples studied. Interestingly, the greatest reduction in mTNF content was observed in 4 LVAD treated patients that had the device explanted becasue cardiac function recovered (78% vs 47%, p<0.045). In addition, diastolic function (dP/dV) improved in 14/17 HOCM patients. These data demonstrate: 1)that mTNF concentrations are reduced by elimination of volume or pressure overload and, 2)that reductions in mTNF content are associated with recovery of cardiac function in DCM patients and improvements in diastolic function in HOCM patients. The findings of the present study demonstrate that changes in volume and pressure regulate mTNF production in human myocardium and suggest that mTNF plays an important role in the regulation of cardiac function. Furthermore, since improvements in systolic and diastolic function were associated with reductions in mTNF concentrations, these data also supports therapeutic strategies aimed to antagonize the biological effects of TNF in cardiac disease states, characterized by volume or pressure overload.
ls
Y3
Y4
Human Cdc5 Controls Mitotic Entry through Specific DNA-Protein Interactions and Is Regulated through a Mitogen-Activated Pathway Harold S. Bernstein; Cardiovascular Research Institute, University of California, San Francisco, CA
Reprogramming of Dysfunctional Gene Expression by Mechanical Unloading in Failing Human Heart Christophe Depre, Gregory L. Shipley, Qiuying Han, O. Howard Frazier, Peter J.A. Davies, Heinrich Taegtmeyer; University of Texas Houston Medical School and Texas Heart Institute, Houston, TX
Each year, one million people in the U.S. experience an acute myocardial infarction; half succumb to the sequelae of myocardial failure. The economic impact of this disease will likely escalate as our ability to treat the acute event improves. Efforts toward myocardial regeneration have been limited by the inability of adult cardiac myocytes to enter mitosis. Expression of transcription factors active during G1/S can induce DNA replication, however, cells thereafter arrest in G2. In eukaryotes, G2/M requires the coordinated expression of many genes, however, mechanisms controlling their transcription are unknown. We recently cloned human Cdc5 (hCdc5), and have shown that it positively regulates G2/M in manunalian cells, hCdc5 contains a DNA binding (DB) domain similar to that seen in Myb, however, its binding specificity is distinct. Upon serum stimulation, hCdc5 is phosphorylated and tcanslocates to the nucleus. While hCdc5 has a GUM-specific effect, it's constitutively expressed in dividing cells, suggesting that its activity in G2 is regulated through post-translational events. We now demonstrate, using mammalian and yeast reporter assays, that hCdc5 contains a transcriptional activation domain, as well as a negative regulatory domain. This confirms hCdc5 as the first transcriptional regulator of G2/M in mammaliancells and suggests intramolecular mechanisms that may control its function. We also show that several domains of hCdc5 are specifically phosphorylated by protein kinase C in vitro. This provides the first evidence that hCdc5 may he regulated by a mitogenactivated pathway. In order to identify human genoIuic sequences that bind hCdc5, we developed a reporter screen in yeast. Four genomic clones sharing a unique 500bp region of homology were isolated. To determine whether these contained hCdc5specific binding sites, we performed amplification and selection of targets from a pool of random oligonucleotides. This identified a pallindromic consensus sequence that binds hCdc5 through its DB domain, as confirmed by gel shift assay. We have demonstrated that hCdc5 can form homodimers through its amino terminus, consistent with its recognition of a pallindromic binding site. This sequence also was found within the homologous region of the genomic clones isolated by our screen. These observations indicate that hCdc5 is capable of site-specific binding, and that one such sequence is present at low frequency in the human genome. Our results have significantly advanced our understanding of cell division, and ulthnately may provide reagents with which to manipulate the cell cycle in non-dividing myocytes.
An unexpected consequence of the management of patients with advanced heart failure is an improvement in cardiac function with mechanical unloading, but the molecular mechanism remains unknown. Here, we investigated the transcriptional adaptation of the failing heart in patients awaiting cardiac transplantation and treated with a left ventricular assist device (LVAD). Transcripts were measured by quantitative PCR on a myocardial sample obtained at the time of LVAD implantation. A second sample from the same heart was taken at the time of LVAD explantation. Compared to controls, myocardium from failing hearts was characterized by a 50-fold increase of TNFe~ and a 10-fold increase of inducible NO synthase (iNOS), two markers of ventricular dysfunction (P<0.001). Transcripts coding for enzymes regulating energy supply (glucose transporters GLUT1 and GLUT4, CPT I) and energy utilization (aMHC, ~MHC, Ca++-ATPase) significantly decreased by 30-60%. The protooncogene c-fos, a marker of transcriptional stimulation, decreased by 90% (P<0.001). In samples from same hearts taken 102 -+ 11 days after mechanical unloading, both TNFa and iNOS had returned to normal. Transcripts coding for both glucose transporters and myosin heavy chain were increased up to 5-fold, with an isoform specificity for the "fetal isoforms" GLUTI and ~MHC. The transcript coding for c-fos was increased 5-fold. Transcripts coding for "housekeeping" proteins were comparable between all groups. These results show that decreased mechanical stress by ventricular unloading significantly reverses the dysfunctional gene expression in the failing human heart in vivo.
•
1
Young Investigator Awards Y1
Y2
Restoration of Contractile Function in Isolated Failing Human Ventricular Cardiac Myocytes by Gene Transfer of SERCA2a Federica del Monte, Sian Harding, Judith K. Gwathmey, Anthony Rosenzweig, Roger J. Hajjar; Cardiov. Res. Center, Mass. Gen. Hosp, Boston Univ., Boston, MA, Imperial College, London
Regulation of Tumor Necrosis Factor by Volume and Pressure Overload in Human Myocardium: Effect of Chronic Ventricular Unloading and Pressure Overload Elimination Guillermo Torre-Amione, Sonny S. Stetson, Keith A. Youker, Jean-Bernard Durand, Sherif F. Nagueh, William H. Spencer, III, Mark L. Entman; Cardiology, Baylor College of Medicine, Houston, TX
Myocardial cells from failing (F) human hearts are characterized by abnormal Ca2+ handling, a negative force frequency relationship (FFR), and decreased sarcoplasmic reticulum (SR) ATPase activity. We have previously shown that overexpmssion of SERCA2a by adenoviral gene transfer increases SR ATPase activity and enhances Ca2+ handling both in vitro and in vivo. To test whether contractile function can be restored by overexpression of SERCA2a in human hearts, myocardial cells isolated from 10 patients with end-stage heart failure were infected with adenoviruses encoding either the SERCA2a gene or reporter genes green fluorescent protein (GFP) and /3-galactosidase for 24 hrs. Cells isolated from 2 non-failing hearts were used as controls (C). As shown below, compared to C myocytes, F myocardial cells had prolonged contraction and calcium transient detected by Fura-2. Overexpression of SERCA2a in F myocytes decreased the time course of both contractions and Caz+ by 19+2% and 27-+4%. F myocytes had decreased Ca2+ release and an increase in diastolic Ca 2+ with increasing frequency (0.1-1.5Hz). Overexpression of SERCA2a restored the FFR in the F cells to normal: increasing frequency resulted in enhanced SR Ca 2+ release and contraction. These results show that gene transfer of SERCA2a improves the contractile function in F human myocytes. Targeting SERCA2a may provide therapeutic benefits in heart failure. Non F a i l i n g
Failing - GFP
Failing - SERCA
An increasing number of observations suggest that TNF plays a pathogenetic role in heart failure; however, the mechanisms that regulate myocardial (m) TNF production in humans are not known. Under experimental conditions, volume and pressure overload stimulate mTNF production; accordingly, the goal of our studies was to determine the effect of changes in ventricular volume and pressure on mTNF expression and its relationship to cardiac function in men. To accomplish this, mTNF concentrations were determined in two distinct patient populations. First, in LV samples from patients with dialated cardiomyopathy(DCM) before and after LVAD implantation, n - 8(volume model). Second, in RV septal biopsies obtained from patients with hypertorphic obstructive cardiomyopathy(HOCM) before and after pressure gradient elimination by ethanol induced septal ablation, n=17(pressure model). TNF content was determined by immunohistochemistry and by semi-quantitative analysis of stained areas. LVAD treated patients had a 42% ± 9% reduction in mTNF concentration after chronic ventricular unloading with LVADs, whereas in HOCM patients, mTNF concentration was reduced to 49.5%-+14% after pressure gradient elimination. We found decreased mTNF content in all 25 paired myocardial samples studied. Interestingly, the greatest reduction in mTNF content was observed in 4 LVAD treated patients that had the device explanted becasue cardiac function recovered (78% vs 47%, p<0.045). In addition, diastolic function (dP/dV) improved in 14/17 HOCM patients. These data demonstrate: 1)that mTNF concentrations are reduced by elimination of volume or pressure overload and, 2)that reductions in mTNF content are associated with recovery of cardiac function in DCM patients and improvements in diastolic function in HOCM patients. The findings of the present study demonstrate that changes in volume and pressure regulate mTNF production in human myocardium and suggest that mTNF plays an important role in the regulation of cardiac function. Furthermore, since improvements in systolic and diastolic function were associated with reductions in mTNF concentrations, these data also supports therapeutic strategies aimed to antagonize the biological effects of TNF in cardiac disease states, characterized by volume or pressure overload.
ls
Y3
Y4
Human Cdc5 Controls Mitotic Entry through Specific DNA-Protein Interactions and Is Regulated through a Mitogen-Activated Pathway Harold S. Bernstein; Cardiovascular Research Institute, University of California, San Francisco, CA
Reprogramming of Dysfunctional Gene Expression by Mechanical Unloading in Failing Human Heart Christophe Depre, Gregory L. Shipley, Qiuying Han, O. Howard Frazier, Peter J.A. Davies, Heinrich Taegtmeyer; University of Texas Houston Medical School and Texas Heart Institute, Houston, TX
Each year, one million people in the U.S. experience an acute myocardial infarction; half succumb to the sequelae of myocardial failure. The economic impact of this disease will likely escalate as our ability to treat the acute event improves. Efforts toward myocardial regeneration have been limited by the inability of adult cardiac myocytes to enter mitosis. Expression of transcription factors active during G1/S can induce DNA replication, however, cells thereafter arrest in G2. In eukaryotes, G2/M requires the coordinated expression of many genes, however, mechanisms controlling their transcription are unknown. We recently cloned human Cdc5 (hCdc5), and have shown that it positively regulates G2/M in manunalian cells, hCdc5 contains a DNA binding (DB) domain similar to that seen in Myb, however, its binding specificity is distinct. Upon serum stimulation, hCdc5 is phosphorylated and tcanslocates to the nucleus. While hCdc5 has a GUM-specific effect, it's constitutively expressed in dividing cells, suggesting that its activity in G2 is regulated through post-translational events. We now demonstrate, using mammalian and yeast reporter assays, that hCdc5 contains a transcriptional activation domain, as well as a negative regulatory domain. This confirms hCdc5 as the first transcriptional regulator of G2/M in mammaliancells and suggests intramolecular mechanisms that may control its function. We also show that several domains of hCdc5 are specifically phosphorylated by protein kinase C in vitro. This provides the first evidence that hCdc5 may he regulated by a mitogenactivated pathway. In order to identify human genoIuic sequences that bind hCdc5, we developed a reporter screen in yeast. Four genomic clones sharing a unique 500bp region of homology were isolated. To determine whether these contained hCdc5specific binding sites, we performed amplification and selection of targets from a pool of random oligonucleotides. This identified a pallindromic consensus sequence that binds hCdc5 through its DB domain, as confirmed by gel shift assay. We have demonstrated that hCdc5 can form homodimers through its amino terminus, consistent with its recognition of a pallindromic binding site. This sequence also was found within the homologous region of the genomic clones isolated by our screen. These observations indicate that hCdc5 is capable of site-specific binding, and that one such sequence is present at low frequency in the human genome. Our results have significantly advanced our understanding of cell division, and ulthnately may provide reagents with which to manipulate the cell cycle in non-dividing myocytes.
An unexpected consequence of the management of patients with advanced heart failure is an improvement in cardiac function with mechanical unloading, but the molecular mechanism remains unknown. Here, we investigated the transcriptional adaptation of the failing heart in patients awaiting cardiac transplantation and treated with a left ventricular assist device (LVAD). Transcripts were measured by quantitative PCR on a myocardial sample obtained at the time of LVAD implantation. A second sample from the same heart was taken at the time of LVAD explantation. Compared to controls, myocardium from failing hearts was characterized by a 50-fold increase of TNFe~ and a 10-fold increase of inducible NO synthase (iNOS), two markers of ventricular dysfunction (P<0.001). Transcripts coding for enzymes regulating energy supply (glucose transporters GLUT1 and GLUT4, CPT I) and energy utilization (aMHC, ~MHC, Ca++-ATPase) significantly decreased by 30-60%. The protooncogene c-fos, a marker of transcriptional stimulation, decreased by 90% (P<0.001). In samples from same hearts taken 102 -+ 11 days after mechanical unloading, both TNFa and iNOS had returned to normal. Transcripts coding for both glucose transporters and myosin heavy chain were increased up to 5-fold, with an isoform specificity for the "fetal isoforms" GLUTI and ~MHC. The transcript coding for c-fos was increased 5-fold. Transcripts coding for "housekeeping" proteins were comparable between all groups. These results show that decreased mechanical stress by ventricular unloading significantly reverses the dysfunctional gene expression in the failing human heart in vivo.