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Unexpected Role of Adenosine-Monophosphate Activated Protein Kinase in Doxorubicin Cardiotoxicity and Metformin-Mediated Cardioprotection
S2 Journal of Cardiac Failure Vol. 25 No. 8S August 2019
JNCNI Basic Science Award Session 001 Intratracheal SERCA2a Gene Therapy Interrupts the Formation of Plexiform Lesions and Rescues from Heart Failure in Severe Pulmonary Arterial Hypertension Anthony S. Fargnoli, Michael Katz, Sarah Gubara, Charles Bridges, Roger J. Hajjar; Icahn School of Medicine at Mt. Sinai, New York, NY Introduction: Drugs fail to improve long term outcomes in pulmonary hypertension (PAH), especially survival in end stages. Severe PAH presents with deadly high pulmonary vascular resistance [PVR] due to the progression of irreversible plexiform lesions [PLX], resulting in heart failure [HF]. SERCA2a [S2A], a calcium handling protein, has been shown effective in PAH via reprogramming endothelial and vascular smooth muscle cells via improving pulmonary flow and disrupting neointimal proliferation. Preliminary S2A studies have shown efficacy in reducing mean PA pressure [mPAP], however in less severe PAH models without HF or PLX. Here, we used a severe surgical PAH HF model and evaluated S2A gene therapy. Hypothesis: S2A gene therapy will reduce PVR and rescue from HF via reducing the degree of PLX. Methods: Twenty rats were divided between control or treatment groups, n=10 ea. All rats were induced with severe PAH via left pneumonectomy combined with Sugen (25mg/kg). Grade 4 maximum plexiform lesions develop with mPAP >35 mmHg @ 3 weeks. AAV1.S2A was administered via intratracheal aerosol at 2.5 £ 1012 VGC after 3 weeks. MRI was performed at baseline, 3 and 6 weeks terminal with invasive mPAP. Harvest assays performed: QPCR of AAV1.S2A genome copies, S2A expression, and PLX pathology. Results: S2A rats survived at 80% compared to control 50%. QPCR detection was very high in 5 S2A animals [1314§858 GC/cell], remaining 3 with average <1 GC/cell. No S2A copies were found in the heart. At 6 weeks: mPAP was reduced in the S2A [41§4] vs. [58§1] mmHg, p<0.05. RV stroke volume greater in S2A [306§ 28] vs [287§21] uL. RV dilatation was reduced in S2A [7.5§0.3] vs. [8.4§0.1] mm, p<0.05. Corresponding PVR was halved in S2A [0.37§0.06] vs [0.71§0.14] mmHg min/mL, p<0.05. S2A expression >2.5 fold in pulmonary smooth muscle anatomy 1B. Unlike control, which presented with higher frequency Grade 4 PLX 1C, the majority of S2A specimens had Grade 1-3 with less occurrence of PLX 1D. Conclusion: S2A gene therapy is effective in reducing the progression of PLX. Gene therapy may serve as an adjunctive option for severe PAH patients.
002 Right Ventricular Sarcomere Dysfunction Underlies Right-Sided Hemodynamic Decompensation in Pulmonary Hypertension Secondary to End-Stage Human Heart Failure Mohammed I. Aslam1, Brian Lin1, Kenneth Bedi2, Kenneth B. Margulies2, David A. Kass1, Steven Hsu1; 1Johns Hopkins Hospital, Baltimore, MD; 2University of Pennsylvania, Philadelphia, PA Background: Right ventricular dysfunction (RVD) drives morbidity and mortality in heart failure with reduced ejection fraction (HFrEF) complicated by secondary pulmonary hypertension (PH). Little is known about RV myofilament function in human HFrEF. We sought to characterize the sarcomeric and molecular phenotype of RV myofilaments in human HFrEF-PH and assess their relation to right-sided hemodynamic parameters of importance. Methods and Results: RV septal tissue was flash frozen from explanted hearts of HFrEF (n=17) and non-failing controls (n=6), and corresponding clinical data were documented. Skinned myocytes were isolated and affixed to a force-length transducer to assess RV myofilament force-calcium curves. RV myofilament maximal calcium-activated force generation (Fmax) was significantly diminished in HFrEF versus control (15.7§4.4 vs. 26.8§5.9 mN/mm2, p<0.0001). Univariate linear regression revealed significant correlations between RV Fmax and pulmonary vascular load (pulmonary vascular resistance, r=0.59, p=0.01; pulmonary arterial compliance, r=-0.53, p=0.03) as well as right heart accommodation to load (log of pulmonary arterial pulsatility index, or log(PAPi), r=0.58, p=0.015; RA pressure, r=-0.54, p=0.025; RA/pulmonary capillary wedge pressure
ratio, r=-0.66, p=0.004). Low PAPi (<1.85) was used to define a subgroup of HFrEF with RVD (n=7, designated HFrEF-RVD). RV Fmax in HFrEF-RVD was significantly lower than HFrEF without RVD (12.3§1.3 vs. 18.2§1.1 mN/mm2, p<0.03) as well as control (p<0.0001, see Figure). Immunoblots revealed a disproportionate decrease in troponin I and tropomyosin phosphorylation, along with upregulation of troponin T, tropomyosin and troponin I in HFrEF-RVD versus both controls and HFrEF without RVD. A direct-acting sarcomere agent (EMD-57033), which stabilizes actin-myosin interaction and acts as a calcium sensitizer, significantly improved RV myocyte Fmax and EC50 in HFrEF-RVD. Conclusions: RV myofilaments demonstrate depressed Fmax in HFrEF-PH. RV sarcomere Fmax is tightly coupled, in opposing fashion, to pulmonary vascular load and right heart accommodation to load. Low PAPi predicts a subgroup of RV myofilaments with a particularly poor RV Fmax, with corresponding and possibly explanatory sarcomeric defects. A sarcomere agent was able to partially restore RV Fmax and increase calcium sensitivity ex vivo and may present a novel mechanism for treating RVD in HFrEF.
003 Unexpected Role of Adenosine-Monophosphate Activated Protein Kinase in Doxorubicin Cardiotoxicity and Metformin-Mediated Cardioprotection Jaclyn Del Pozzo, Puja Mehta, Fei Cai, Cairong Li, Satoru Kobayashi, Qiangrong Liang; New York Institute of Technology College of Osteopathic Medicine, Old Westbury, NY Doxorubicin (DOX), a commonly used antineoplastic drug, raises clinical concern due to its irreversible cardiotoxicity. Metformin (MET), a drug used for the treatment of type 2 diabetes mellitus, has been suggested as a cardioprotective agent against DOX-induced cardiotoxicity. However, the underlying mechanisms are not fully understood. Recent studies have suggested that Adenosine-monophosphate activated protein kinase (AMPK) may be responsible for the ability of MET to decrease DOXinduced cardiomyocyte death in culture. In the present study, we determined the role of AMPK pathway in DOX cardiotoxicity and in MET-induced cardioprotection using AMPK knockout mice. Wild type (WT) and AMPK knockout (AKO) mice were distributed into four groups (control, MET, DOX, DOX + MET). Mice were administered with MET (200 mg/kg/d, oral) every day for 6 consecutive days and DOX once only on day 3 (20 mg/kg, i.p.). Echocardiography confirmed that MET was sufficient to reverse DOX-impaired cardiac function in WT mice as shown by fractional shortening (DOX 27.8§0.96%, DOX+MET 34.9§1.79%, and Con 40.0§ 1.28%, n of 7 to 11). Surprisingly, DOX-induced-impairment of cardiac function was not further increased in AMPK knockout mice. Instead, there was a tendency towards reduced cardiac damage in AMPK knockout mice, suggesting that AMPK may contribute to DOX cardiotoxicity, in contrast to our initial hypothesis. As a result, MET did not provide further protection (DOX 33.0§1.23%, DOX+MET 35.7§1.15%, and Con 42.3§1.55%, n of 10 to12). These results were supported by serum levels of lactate dehydrogenase (LDH) and cardiac Troponin I as well as by in vitro studies using cultured cardiomyocytes. In summary, our results supported the concurrent use of MET and DOX to provide cardiac protection for patients that receive antitumor treatment. However, the true role of AMPK in DOX cardiotoxicity needs to be re-evaluated by further studies. Also, the cardioprotective effects of MET may be mediated through signaling pathways unrelated to AMPK.
004 A Multi-Marker Approach to Predict Incident Heart Failure Risk in Chronic Kidney Disease Sadeer Al-Kindi, Mirela Dobre, David Zidar; University Hospitals Cleveland Medical Center, Cleveland, OH Background: Chronic kidney disease (CKD) is associated with increased risk for heart failure (HF). Several pathophysiologic pathways are activated in CKD which may lead to HF. We sought to study the utility of a multi-marker approach to predict HF risk in CKD. Hypothesis: We hypothesized that a multi-marker panel can help identify patients with CKD who are at risk for HF. Methods: We studied 3557 patients with CKD (eGFR < 60 ml/min per 1.73 m2) enrolled in the Chronic Renal Insufficiency Cohort (CRIC) without prevalent HF and who underwent serum/plasma assays for 11 biomarkers at study enrollment (CXCL12, Fractalkine,
JNCNI Basic Science Award Session 001 Intratracheal SERCA2a Gene Therapy Interrupts the Formation of Plexiform Lesions and Rescues from Heart Failure in Severe Pulmonary Arterial Hypertension Anthony S. Fargnoli, Michael Katz, Sarah Gubara, Charles Bridges, Roger J. Hajjar; Icahn School of Medicine at Mt. Sinai, New York, NY Introduction: Drugs fail to improve long term outcomes in pulmonary hypertension (PAH), especially survival in end stages. Severe PAH presents with deadly high pulmonary vascular resistance [PVR] due to the progression of irreversible plexiform lesions [PLX], resulting in heart failure [HF]. SERCA2a [S2A], a calcium handling protein, has been shown effective in PAH via reprogramming endothelial and vascular smooth muscle cells via improving pulmonary flow and disrupting neointimal proliferation. Preliminary S2A studies have shown efficacy in reducing mean PA pressure [mPAP], however in less severe PAH models without HF or PLX. Here, we used a severe surgical PAH HF model and evaluated S2A gene therapy. Hypothesis: S2A gene therapy will reduce PVR and rescue from HF via reducing the degree of PLX. Methods: Twenty rats were divided between control or treatment groups, n=10 ea. All rats were induced with severe PAH via left pneumonectomy combined with Sugen (25mg/kg). Grade 4 maximum plexiform lesions develop with mPAP >35 mmHg @ 3 weeks. AAV1.S2A was administered via intratracheal aerosol at 2.5 £ 1012 VGC after 3 weeks. MRI was performed at baseline, 3 and 6 weeks terminal with invasive mPAP. Harvest assays performed: QPCR of AAV1.S2A genome copies, S2A expression, and PLX pathology. Results: S2A rats survived at 80% compared to control 50%. QPCR detection was very high in 5 S2A animals [1314§858 GC/cell], remaining 3 with average <1 GC/cell. No S2A copies were found in the heart. At 6 weeks: mPAP was reduced in the S2A [41§4] vs. [58§1] mmHg, p<0.05. RV stroke volume greater in S2A [306§ 28] vs [287§21] uL. RV dilatation was reduced in S2A [7.5§0.3] vs. [8.4§0.1] mm, p<0.05. Corresponding PVR was halved in S2A [0.37§0.06] vs [0.71§0.14] mmHg min/mL, p<0.05. S2A expression >2.5 fold in pulmonary smooth muscle anatomy 1B. Unlike control, which presented with higher frequency Grade 4 PLX 1C, the majority of S2A specimens had Grade 1-3 with less occurrence of PLX 1D. Conclusion: S2A gene therapy is effective in reducing the progression of PLX. Gene therapy may serve as an adjunctive option for severe PAH patients.
002 Right Ventricular Sarcomere Dysfunction Underlies Right-Sided Hemodynamic Decompensation in Pulmonary Hypertension Secondary to End-Stage Human Heart Failure Mohammed I. Aslam1, Brian Lin1, Kenneth Bedi2, Kenneth B. Margulies2, David A. Kass1, Steven Hsu1; 1Johns Hopkins Hospital, Baltimore, MD; 2University of Pennsylvania, Philadelphia, PA Background: Right ventricular dysfunction (RVD) drives morbidity and mortality in heart failure with reduced ejection fraction (HFrEF) complicated by secondary pulmonary hypertension (PH). Little is known about RV myofilament function in human HFrEF. We sought to characterize the sarcomeric and molecular phenotype of RV myofilaments in human HFrEF-PH and assess their relation to right-sided hemodynamic parameters of importance. Methods and Results: RV septal tissue was flash frozen from explanted hearts of HFrEF (n=17) and non-failing controls (n=6), and corresponding clinical data were documented. Skinned myocytes were isolated and affixed to a force-length transducer to assess RV myofilament force-calcium curves. RV myofilament maximal calcium-activated force generation (Fmax) was significantly diminished in HFrEF versus control (15.7§4.4 vs. 26.8§5.9 mN/mm2, p<0.0001). Univariate linear regression revealed significant correlations between RV Fmax and pulmonary vascular load (pulmonary vascular resistance, r=0.59, p=0.01; pulmonary arterial compliance, r=-0.53, p=0.03) as well as right heart accommodation to load (log of pulmonary arterial pulsatility index, or log(PAPi), r=0.58, p=0.015; RA pressure, r=-0.54, p=0.025; RA/pulmonary capillary wedge pressure
ratio, r=-0.66, p=0.004). Low PAPi (<1.85) was used to define a subgroup of HFrEF with RVD (n=7, designated HFrEF-RVD). RV Fmax in HFrEF-RVD was significantly lower than HFrEF without RVD (12.3§1.3 vs. 18.2§1.1 mN/mm2, p<0.03) as well as control (p<0.0001, see Figure). Immunoblots revealed a disproportionate decrease in troponin I and tropomyosin phosphorylation, along with upregulation of troponin T, tropomyosin and troponin I in HFrEF-RVD versus both controls and HFrEF without RVD. A direct-acting sarcomere agent (EMD-57033), which stabilizes actin-myosin interaction and acts as a calcium sensitizer, significantly improved RV myocyte Fmax and EC50 in HFrEF-RVD. Conclusions: RV myofilaments demonstrate depressed Fmax in HFrEF-PH. RV sarcomere Fmax is tightly coupled, in opposing fashion, to pulmonary vascular load and right heart accommodation to load. Low PAPi predicts a subgroup of RV myofilaments with a particularly poor RV Fmax, with corresponding and possibly explanatory sarcomeric defects. A sarcomere agent was able to partially restore RV Fmax and increase calcium sensitivity ex vivo and may present a novel mechanism for treating RVD in HFrEF.
003 Unexpected Role of Adenosine-Monophosphate Activated Protein Kinase in Doxorubicin Cardiotoxicity and Metformin-Mediated Cardioprotection Jaclyn Del Pozzo, Puja Mehta, Fei Cai, Cairong Li, Satoru Kobayashi, Qiangrong Liang; New York Institute of Technology College of Osteopathic Medicine, Old Westbury, NY Doxorubicin (DOX), a commonly used antineoplastic drug, raises clinical concern due to its irreversible cardiotoxicity. Metformin (MET), a drug used for the treatment of type 2 diabetes mellitus, has been suggested as a cardioprotective agent against DOX-induced cardiotoxicity. However, the underlying mechanisms are not fully understood. Recent studies have suggested that Adenosine-monophosphate activated protein kinase (AMPK) may be responsible for the ability of MET to decrease DOXinduced cardiomyocyte death in culture. In the present study, we determined the role of AMPK pathway in DOX cardiotoxicity and in MET-induced cardioprotection using AMPK knockout mice. Wild type (WT) and AMPK knockout (AKO) mice were distributed into four groups (control, MET, DOX, DOX + MET). Mice were administered with MET (200 mg/kg/d, oral) every day for 6 consecutive days and DOX once only on day 3 (20 mg/kg, i.p.). Echocardiography confirmed that MET was sufficient to reverse DOX-impaired cardiac function in WT mice as shown by fractional shortening (DOX 27.8§0.96%, DOX+MET 34.9§1.79%, and Con 40.0§ 1.28%, n of 7 to 11). Surprisingly, DOX-induced-impairment of cardiac function was not further increased in AMPK knockout mice. Instead, there was a tendency towards reduced cardiac damage in AMPK knockout mice, suggesting that AMPK may contribute to DOX cardiotoxicity, in contrast to our initial hypothesis. As a result, MET did not provide further protection (DOX 33.0§1.23%, DOX+MET 35.7§1.15%, and Con 42.3§1.55%, n of 10 to12). These results were supported by serum levels of lactate dehydrogenase (LDH) and cardiac Troponin I as well as by in vitro studies using cultured cardiomyocytes. In summary, our results supported the concurrent use of MET and DOX to provide cardiac protection for patients that receive antitumor treatment. However, the true role of AMPK in DOX cardiotoxicity needs to be re-evaluated by further studies. Also, the cardioprotective effects of MET may be mediated through signaling pathways unrelated to AMPK.
004 A Multi-Marker Approach to Predict Incident Heart Failure Risk in Chronic Kidney Disease Sadeer Al-Kindi, Mirela Dobre, David Zidar; University Hospitals Cleveland Medical Center, Cleveland, OH Background: Chronic kidney disease (CKD) is associated with increased risk for heart failure (HF). Several pathophysiologic pathways are activated in CKD which may lead to HF. We sought to study the utility of a multi-marker approach to predict HF risk in CKD. Hypothesis: We hypothesized that a multi-marker panel can help identify patients with CKD who are at risk for HF. Methods: We studied 3557 patients with CKD (eGFR < 60 ml/min per 1.73 m2) enrolled in the Chronic Renal Insufficiency Cohort (CRIC) without prevalent HF and who underwent serum/plasma assays for 11 biomarkers at study enrollment (CXCL12, Fractalkine,