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Evidence of the Presence of Autoantibodies Specific for β1 Adrenergic Receptor in Failing Human Heart Tissue
S30 Journal of Cardiac Failure Vol. 21 No. 8S August 2015
Molecular Biology/Genetics/Cell Therapy I 045 Presence of Endothelial to Mesenchymal Transition in End Stage Human Myocardial Samples-documentation in Human Heart Failure for the First Time Cesar Uribe, Andrea M. Cordero-Reyes, Keith A. Youker, Barry H. Trachtenberg, Guha Ashrith, Jerry D. Estep, Guillermo Torre-Amione, Erik E. Suarez, John P. Cooke, Arvind Bhimaraj; The Methodist Hospital, Houston, TX Background: Endothelial to Mesenchymal Transition (EndMT) and the reverse phenomenon of mesenchymal to endothelial transitioning (MET) has been shown to contribute to fibrosis in rodent models of heart failure. There are no studies documenting the same in human cardiac tissue. Methods: We performed immunofluorescence dual staining on end stage heart failure myocardial samples obtained from our bio repository. Staining for endothelial and mesenchymal markers were performed with Anti-VE-Cadherin (Santa Cruz Biotechnologies) and anti-Fibroblast specific protein 1 (Dako Antibodies) respectively using standard immunofluoresence techniques. Presence of dual staining on fluorescence microscopy was considered as positive for the presence of EndMT. Vascular density was assessed using a DAB (3-3’ Diaminobenzidine) technique with Anti-CD31 (PECAM, Abcam). Results: Of the end stage heart failure samples, 18 were from left ventricular core during LVAD implant and 5 during a direct transplant as status 1A (axillary intraaortic balloon pump). 10 hearts of patients bridged to transplant with a continuous flow LVAD (Days under support 3436228) were also studied. Mean age was 55y with 82% males and 74% white. 70% of all samples had evidence of EndMT. The number of cells ranged from 1 to 5 per high power field. There was no significant difference between patient characteristics between those with evidence of EndMT versus not, except a higher prevalence of diabetes in the latter group. (Fig 1B) 74% of the end-stage heart failure samples and 60% of the post-VAD samples showed the presence of EndMT cells. Vascular Density was higher in the samples exhibiting EndMT phenomenon. (Figure 1A). Conclusion: We have documented for the first time, the existence of endothelial-mesenchymal transition in human end stage myocardial samples corroborating earlier pioneering studies in rodents. Interestingly the existence of such dual staining cells in end stage hearts (which is highly fibrotic) suggests a possible role of ongoing fibrosis with endothelial to mesenchymal transitioning or potential antifibrotic mechanism though mesenchymal to endothelial transitioning. Further studies of the later process will be warranted to explore potential anti-fibrotic therapeutic implications.
been shown to be protective in experimental models of myocardial infarction, persistent activation has been associated to myocardial dysfunction and cardiomyopathy. Methods: Paired myocardial samples from 6 patients (3- pulsatile Novacor LVAD (NVAD); 3- continuous flow DeBakey assist devices (DVAD)) were collected at the time of LVAD implantation and later at the time of transplantation. An affymetrix microarray assay was used to analyze mRNA expression for 54,675 genes in the paired samples. Hypoxia inducible factor and related regulatory genes were studied. Results: Median days on support was 74+/- 53 months; 5 were male, 3 Caucasian, 2 African Americans and 1 Hispanic. All were ischemic in origin. After mechanical circulatory support there was a 78% increase in HIF gene expression from baseline and a 172% increase in CBP/p300 interacting transactivator with ED-rich tail 2 (CITED2). Other important negative regulators of HIF activity such as cellular prolyl-hydroxylases were also increased. (Table 1). Vascular endothelial growth factor, AKT1 and pyruvate kinase expression was increased. Conclusions: After left ventricular support as bridge to transplant there was an increase in the expression of HIF coupled with an increase in the expression CITED2 which may attenuate downstream effects of HIF activation. Vascular proliferation and glucose metabolism were also increased. Further studies are needed to understand the complex regulation of HIF and the effects of type and timing of mechanical support unloading. Table 1.
047 Evidence of the Presence of Autoantibodies Specific for b1 Adrenergic Receptor in Failing Human Heart Tissue Yuji Nagatomo, Christine S. Moravec, W. H. Wilson Tang; Cleveland Clinic, Cleveland, OH
Figure 1.
046 Hypoxia Inducible Factor (HIF) Adaptation After Mechanical Circulatory Unloading of the Heart. A Gene Expression Analysis Study of Paired Myocardial Samples Paulino Alvarez, Cesar Uribe, Andrea M. Cordero-Reyes, Keith A. Youker, Guillermo Torre-Amione, Jerry D. Estep; The Methodist Hospital, Houston, TX Background: Hypoxia-inducible factor (HIF) is a transcription factor that plays a central role in the adaptation to oxygen availability. Although acute activation has
Introduction: Autoimmune disorders may be involved in the pathogenesis of some patients with heart failure (HF). Several anti-cardiac autoantibodies (AAbs) have been reported to be present in sera from patients with dilated cardiomyopathy (DCM) and other etiologies. Among those, AAbs recognizing the b1 adrenergic receptor (b1AR-AAbs) appear to be most well documented and clinically relevant. b1AR-AAbs show agonist-like effects, have some detrimental effects on cardiomyocytes, and induce persistent myocardial damage. Some studies have reported that the elimination of anti-cardiac AAbs belonging to the IgG3 subclass by immunoadsorption was associated with beneficial effects, such as improvement of hemodynamics or LVEF in DCM patients. However, in all of the human studies conducted so far, the b1AR-AAbs have been detected only in serum or plasma, and it has not been shown that this AAb actually can be detected in human cardiac tissue. Objective: The objective of this study was to establish a method for determining whether b1ARAAbs belonging to the IgG or IgG3 subclass are deposited in cardiac tissue of patients with advanced HF. Methods: Failing LV tissue from subjects who underwent cardiac transplantation because of advanced DCM (n58, age 5467, gender male 7, race white 7 black 1) were collected. Three non-failing donor heart samples were used as controls. Frozen heart tissue was embedded in OCT for immunohistochemistry. IgG or IgG3 deposited in LV tissue was stained with FITC-conjugated anti-human IgG or IgG3 antibody. A second piece of LV tissue was homogenized with lysis buffer and IgG deposited in LV tissue was extracted using protein G affinity
The 19th Annual Scientific Meeting chromatography. In order to determine the presence of b1AR-AAb belonging to IgG or IgG3 subclass, purified IgG was applied to ELISA using a 96 well plate coated with the synthetic peptide corresponding to the putative sequence of 2nd extracellular loop domain of human b1AR as the epitope peptide. Results: The IgG deposition in LV tissue was detected in 6 out of 8 DCM subjects (75%) and IgG3 was detected in 3 subjects (38%), whereas none of these were detected in any of the non-failing donor hearts. The presence of IgG extracted from LV tissue was confirmed by Western blot technique. In IgG extracted from LV tissue homogenate IgG-b1AR-AAb was shown to be positive in 4 out of 8 subjects (50%) with DCM and IgG3-b1AR-AAb was positive just in 1 subject (13%) with DCM, whereas none of these were detected in any of the non-failing donor hearts. Conclusions: We demonstrated that b1AR-AAb was present in cardiac tissue in the majority of patients with advanced HF due to DCM. Further investigation will be needed to understand the clinical and prognostic significance of b1AR-AAb detected in failing hearts.
048 Carriers of Variants of Unknown Significance Have Intermediate Hypertrophic Cardiomyopathy Phenotypes Patrick J. Warner, Ashley A. Cronkright, Noreen Dolan, Martin Maron, Gordon S. Huggins; Tufts Medical Center, Boston, MA Introduction: Genetic testing for Hypertrophic Cardiomyopathy (HCM) can identify sarcomere gene mutations that cause HCM (Class I variants) in addition to a variant of unknown significance (VUS/Class II variant) whose relationship to HCM is less well defined. We hypothesized that the PolyPhen protein prediction program can discriminate VUS/Class II variants that likely contribute significantly to HCM from mutations that have a more benign effect. Methods and Results: We performed a retrospective chart review on all patients at the Tufts HCM Center who underwent genetic testing from 2008-2014 (N5227, Age 52.3 6 14.7years, 62.9% male). Of those subjects, 57.7% were mutation negative, 20.2% had VUS/Class II mutations, and 22.1% had Class I mutations. Mean maximal left ventricular wall thickness (LVWT) and survival free of surgical intervention, classified as age of myectomy or transplant, was significantly different across the 3 groups (p!0.05). Class I mutation carriers had the greatest LVWT (21.0 6 4.7 mm), followed by VUS/Class II (20.2 6 5.5 mm), and no mutation (18.4 6 4.6mm). Carriers of Class I mutations had significantly greater LVWT and shorter time to surgical intervention compared with those that were mutation negative (both p!0.05). By comparison, the maximal LVWT and time to surgical intervention in VUS/Class II carriers was between that of Class I mutation carriers and HCM patients in whom no mutation was found. The PolyPhen score for the VUS/Class II mutations identified in our cohort ranged from 0 (benign) to 1 (probably damaging); mean score was 0.75 6 0.38. A bimodal distribution appeared to be present with PolyPhen scores clustered below 0.2 (suggesting more benign) or above 0.8 (suggesting more deleterious). Univariate analysis demonstrated a borderline significant linear relationship between the VUS/Class II PolyPhen score and maximal LVWT (R50.267, p50.087); there was no relationship between PolyPhen score and time to surgical intervention (R5-0.041, p50.900). Conclusion: In summary, our data demonstrate that VUS/Class II mutation carriers with HCM have an intermediary degree of maximal LVWT and survival free of surgical intervention between Class I mutation carriers and HCM patients in whom no mutation was identified. The PolyPhen protein prediction score did not discriminate a subset of VUS/Class II mutations that were associated with increased LVWT or the need for septal reduction surgery. Further development of protein prediction methods is required to better predict clinical outcomes in an HCM patient carrying a VUS/ Class II mutation.
049 Neuregulin/ERBB Signaling in Human Ventricular Myocardial Progenitor Cells Sergey Ryzhov1, Oleg Tikhomirov1, Rutwik Rath1, Douglas B. Sawyer1,2; 1Maine Medical Center Research Institute, Scarborough, ME; 2Maine Medical Center, Portland, ME Introduction: Endogenous cardiac progenitor cells have been characterized by their ability to differentiate into cardiac myocytes, although at very low levels. These cells also give rise to other cardiac cell types, including endothelial cells, fibroblasts and myofibroblasts. Directing differentiation of progenitors toward cardiac myocytes and limiting their conversion into non-myocyte cells, specifically fibroblasts/myofibroblasts could be a successful approach to regenerate cardiac tissue after injury. Methods and Results: We have previously reported that neuregulin (NRG) promotes myogenic differentiation of murine embryonic stem cells via induction of Nkx2.5 and cardiac troponin T. The induction of myogenic differentiation was dependent upon expression of ERBB3 receptors. We sought evidence that adult myocardium has similar progenitor cell populations. In adult mice, Sca-1 and CD105 positive cells, located adjacent to the basal lamina and in proximity with endothelial cells, were characterized as cardiac progenitors. Additional characteristics of this population are low or negative expression of CD31/PECAM1 and absence of CD34 and CD45 hematopoietic cell markers. No expression of c-Kit was found on these cells. We found that these cells can differentiate to multiple lineages, including myofibroblasts which is suppressed by NRG treatment. Immunohistochemical analysis revealed that expression of ERBB2 and ERBB3 in human heart is associated with vascular/peri-vascular regions, well characterized structural component of the cardiac stem cell niche. Using single-cell clonogenic technique we isolated human cardiac
HFSA
S31
progenitors. These cells were characterized by cell surface expression of CD105 and absence of CD31 endothelial marker, CD45 and CD117/c-Kit hematopoietic markers. We found that these cells express both ERBB2 and ERBB3 but not EGFR or ERBB4 receptors. Using specific antibodies against ERBB receptors we found that ERBB2 and ERBB3 receptors are present on the surface of human cardiac progenitors. We found no expression of ERBB receptors in c-Kit positive cells, indicating that neuregulin/ERBB signaling is exclusively linked to the regulation of CD105pos CD31neg cardiac progenitors. Conclusion: Thus, our study identified ERBB2 and ERBB3 receptors on cardiac stromal cells as potential targets for neuregulin-dependent induction of heart regeneration. Support: (U01 HL100398).
Molecular Biology/Genetics/Cell Therapy I 045 Presence of Endothelial to Mesenchymal Transition in End Stage Human Myocardial Samples-documentation in Human Heart Failure for the First Time Cesar Uribe, Andrea M. Cordero-Reyes, Keith A. Youker, Barry H. Trachtenberg, Guha Ashrith, Jerry D. Estep, Guillermo Torre-Amione, Erik E. Suarez, John P. Cooke, Arvind Bhimaraj; The Methodist Hospital, Houston, TX Background: Endothelial to Mesenchymal Transition (EndMT) and the reverse phenomenon of mesenchymal to endothelial transitioning (MET) has been shown to contribute to fibrosis in rodent models of heart failure. There are no studies documenting the same in human cardiac tissue. Methods: We performed immunofluorescence dual staining on end stage heart failure myocardial samples obtained from our bio repository. Staining for endothelial and mesenchymal markers were performed with Anti-VE-Cadherin (Santa Cruz Biotechnologies) and anti-Fibroblast specific protein 1 (Dako Antibodies) respectively using standard immunofluoresence techniques. Presence of dual staining on fluorescence microscopy was considered as positive for the presence of EndMT. Vascular density was assessed using a DAB (3-3’ Diaminobenzidine) technique with Anti-CD31 (PECAM, Abcam). Results: Of the end stage heart failure samples, 18 were from left ventricular core during LVAD implant and 5 during a direct transplant as status 1A (axillary intraaortic balloon pump). 10 hearts of patients bridged to transplant with a continuous flow LVAD (Days under support 3436228) were also studied. Mean age was 55y with 82% males and 74% white. 70% of all samples had evidence of EndMT. The number of cells ranged from 1 to 5 per high power field. There was no significant difference between patient characteristics between those with evidence of EndMT versus not, except a higher prevalence of diabetes in the latter group. (Fig 1B) 74% of the end-stage heart failure samples and 60% of the post-VAD samples showed the presence of EndMT cells. Vascular Density was higher in the samples exhibiting EndMT phenomenon. (Figure 1A). Conclusion: We have documented for the first time, the existence of endothelial-mesenchymal transition in human end stage myocardial samples corroborating earlier pioneering studies in rodents. Interestingly the existence of such dual staining cells in end stage hearts (which is highly fibrotic) suggests a possible role of ongoing fibrosis with endothelial to mesenchymal transitioning or potential antifibrotic mechanism though mesenchymal to endothelial transitioning. Further studies of the later process will be warranted to explore potential anti-fibrotic therapeutic implications.
been shown to be protective in experimental models of myocardial infarction, persistent activation has been associated to myocardial dysfunction and cardiomyopathy. Methods: Paired myocardial samples from 6 patients (3- pulsatile Novacor LVAD (NVAD); 3- continuous flow DeBakey assist devices (DVAD)) were collected at the time of LVAD implantation and later at the time of transplantation. An affymetrix microarray assay was used to analyze mRNA expression for 54,675 genes in the paired samples. Hypoxia inducible factor and related regulatory genes were studied. Results: Median days on support was 74+/- 53 months; 5 were male, 3 Caucasian, 2 African Americans and 1 Hispanic. All were ischemic in origin. After mechanical circulatory support there was a 78% increase in HIF gene expression from baseline and a 172% increase in CBP/p300 interacting transactivator with ED-rich tail 2 (CITED2). Other important negative regulators of HIF activity such as cellular prolyl-hydroxylases were also increased. (Table 1). Vascular endothelial growth factor, AKT1 and pyruvate kinase expression was increased. Conclusions: After left ventricular support as bridge to transplant there was an increase in the expression of HIF coupled with an increase in the expression CITED2 which may attenuate downstream effects of HIF activation. Vascular proliferation and glucose metabolism were also increased. Further studies are needed to understand the complex regulation of HIF and the effects of type and timing of mechanical support unloading. Table 1.
047 Evidence of the Presence of Autoantibodies Specific for b1 Adrenergic Receptor in Failing Human Heart Tissue Yuji Nagatomo, Christine S. Moravec, W. H. Wilson Tang; Cleveland Clinic, Cleveland, OH
Figure 1.
046 Hypoxia Inducible Factor (HIF) Adaptation After Mechanical Circulatory Unloading of the Heart. A Gene Expression Analysis Study of Paired Myocardial Samples Paulino Alvarez, Cesar Uribe, Andrea M. Cordero-Reyes, Keith A. Youker, Guillermo Torre-Amione, Jerry D. Estep; The Methodist Hospital, Houston, TX Background: Hypoxia-inducible factor (HIF) is a transcription factor that plays a central role in the adaptation to oxygen availability. Although acute activation has
Introduction: Autoimmune disorders may be involved in the pathogenesis of some patients with heart failure (HF). Several anti-cardiac autoantibodies (AAbs) have been reported to be present in sera from patients with dilated cardiomyopathy (DCM) and other etiologies. Among those, AAbs recognizing the b1 adrenergic receptor (b1AR-AAbs) appear to be most well documented and clinically relevant. b1AR-AAbs show agonist-like effects, have some detrimental effects on cardiomyocytes, and induce persistent myocardial damage. Some studies have reported that the elimination of anti-cardiac AAbs belonging to the IgG3 subclass by immunoadsorption was associated with beneficial effects, such as improvement of hemodynamics or LVEF in DCM patients. However, in all of the human studies conducted so far, the b1AR-AAbs have been detected only in serum or plasma, and it has not been shown that this AAb actually can be detected in human cardiac tissue. Objective: The objective of this study was to establish a method for determining whether b1ARAAbs belonging to the IgG or IgG3 subclass are deposited in cardiac tissue of patients with advanced HF. Methods: Failing LV tissue from subjects who underwent cardiac transplantation because of advanced DCM (n58, age 5467, gender male 7, race white 7 black 1) were collected. Three non-failing donor heart samples were used as controls. Frozen heart tissue was embedded in OCT for immunohistochemistry. IgG or IgG3 deposited in LV tissue was stained with FITC-conjugated anti-human IgG or IgG3 antibody. A second piece of LV tissue was homogenized with lysis buffer and IgG deposited in LV tissue was extracted using protein G affinity
The 19th Annual Scientific Meeting chromatography. In order to determine the presence of b1AR-AAb belonging to IgG or IgG3 subclass, purified IgG was applied to ELISA using a 96 well plate coated with the synthetic peptide corresponding to the putative sequence of 2nd extracellular loop domain of human b1AR as the epitope peptide. Results: The IgG deposition in LV tissue was detected in 6 out of 8 DCM subjects (75%) and IgG3 was detected in 3 subjects (38%), whereas none of these were detected in any of the non-failing donor hearts. The presence of IgG extracted from LV tissue was confirmed by Western blot technique. In IgG extracted from LV tissue homogenate IgG-b1AR-AAb was shown to be positive in 4 out of 8 subjects (50%) with DCM and IgG3-b1AR-AAb was positive just in 1 subject (13%) with DCM, whereas none of these were detected in any of the non-failing donor hearts. Conclusions: We demonstrated that b1AR-AAb was present in cardiac tissue in the majority of patients with advanced HF due to DCM. Further investigation will be needed to understand the clinical and prognostic significance of b1AR-AAb detected in failing hearts.
048 Carriers of Variants of Unknown Significance Have Intermediate Hypertrophic Cardiomyopathy Phenotypes Patrick J. Warner, Ashley A. Cronkright, Noreen Dolan, Martin Maron, Gordon S. Huggins; Tufts Medical Center, Boston, MA Introduction: Genetic testing for Hypertrophic Cardiomyopathy (HCM) can identify sarcomere gene mutations that cause HCM (Class I variants) in addition to a variant of unknown significance (VUS/Class II variant) whose relationship to HCM is less well defined. We hypothesized that the PolyPhen protein prediction program can discriminate VUS/Class II variants that likely contribute significantly to HCM from mutations that have a more benign effect. Methods and Results: We performed a retrospective chart review on all patients at the Tufts HCM Center who underwent genetic testing from 2008-2014 (N5227, Age 52.3 6 14.7years, 62.9% male). Of those subjects, 57.7% were mutation negative, 20.2% had VUS/Class II mutations, and 22.1% had Class I mutations. Mean maximal left ventricular wall thickness (LVWT) and survival free of surgical intervention, classified as age of myectomy or transplant, was significantly different across the 3 groups (p!0.05). Class I mutation carriers had the greatest LVWT (21.0 6 4.7 mm), followed by VUS/Class II (20.2 6 5.5 mm), and no mutation (18.4 6 4.6mm). Carriers of Class I mutations had significantly greater LVWT and shorter time to surgical intervention compared with those that were mutation negative (both p!0.05). By comparison, the maximal LVWT and time to surgical intervention in VUS/Class II carriers was between that of Class I mutation carriers and HCM patients in whom no mutation was found. The PolyPhen score for the VUS/Class II mutations identified in our cohort ranged from 0 (benign) to 1 (probably damaging); mean score was 0.75 6 0.38. A bimodal distribution appeared to be present with PolyPhen scores clustered below 0.2 (suggesting more benign) or above 0.8 (suggesting more deleterious). Univariate analysis demonstrated a borderline significant linear relationship between the VUS/Class II PolyPhen score and maximal LVWT (R50.267, p50.087); there was no relationship between PolyPhen score and time to surgical intervention (R5-0.041, p50.900). Conclusion: In summary, our data demonstrate that VUS/Class II mutation carriers with HCM have an intermediary degree of maximal LVWT and survival free of surgical intervention between Class I mutation carriers and HCM patients in whom no mutation was identified. The PolyPhen protein prediction score did not discriminate a subset of VUS/Class II mutations that were associated with increased LVWT or the need for septal reduction surgery. Further development of protein prediction methods is required to better predict clinical outcomes in an HCM patient carrying a VUS/ Class II mutation.
049 Neuregulin/ERBB Signaling in Human Ventricular Myocardial Progenitor Cells Sergey Ryzhov1, Oleg Tikhomirov1, Rutwik Rath1, Douglas B. Sawyer1,2; 1Maine Medical Center Research Institute, Scarborough, ME; 2Maine Medical Center, Portland, ME Introduction: Endogenous cardiac progenitor cells have been characterized by their ability to differentiate into cardiac myocytes, although at very low levels. These cells also give rise to other cardiac cell types, including endothelial cells, fibroblasts and myofibroblasts. Directing differentiation of progenitors toward cardiac myocytes and limiting their conversion into non-myocyte cells, specifically fibroblasts/myofibroblasts could be a successful approach to regenerate cardiac tissue after injury. Methods and Results: We have previously reported that neuregulin (NRG) promotes myogenic differentiation of murine embryonic stem cells via induction of Nkx2.5 and cardiac troponin T. The induction of myogenic differentiation was dependent upon expression of ERBB3 receptors. We sought evidence that adult myocardium has similar progenitor cell populations. In adult mice, Sca-1 and CD105 positive cells, located adjacent to the basal lamina and in proximity with endothelial cells, were characterized as cardiac progenitors. Additional characteristics of this population are low or negative expression of CD31/PECAM1 and absence of CD34 and CD45 hematopoietic cell markers. No expression of c-Kit was found on these cells. We found that these cells can differentiate to multiple lineages, including myofibroblasts which is suppressed by NRG treatment. Immunohistochemical analysis revealed that expression of ERBB2 and ERBB3 in human heart is associated with vascular/peri-vascular regions, well characterized structural component of the cardiac stem cell niche. Using single-cell clonogenic technique we isolated human cardiac
HFSA
S31
progenitors. These cells were characterized by cell surface expression of CD105 and absence of CD31 endothelial marker, CD45 and CD117/c-Kit hematopoietic markers. We found that these cells express both ERBB2 and ERBB3 but not EGFR or ERBB4 receptors. Using specific antibodies against ERBB receptors we found that ERBB2 and ERBB3 receptors are present on the surface of human cardiac progenitors. We found no expression of ERBB receptors in c-Kit positive cells, indicating that neuregulin/ERBB signaling is exclusively linked to the regulation of CD105pos CD31neg cardiac progenitors. Conclusion: Thus, our study identified ERBB2 and ERBB3 receptors on cardiac stromal cells as potential targets for neuregulin-dependent induction of heart regeneration. Support: (U01 HL100398).