Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking induced RV fibrosis. Here we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the LV. In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/ epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Further, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared to wild-type controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a novel therapeutic strategy in the setting of increased RV afterload.
Alexander Vang, Denielli da Silva Gonçalves Bos, Ana Fernandez-Nicolas, Peng Zhang, Alan R. Morrison, Thomas J. Mancini, Richard T. Clements, Iuliia Polina, Michael W. Cypress, Bong Sook Jhun, Edward Hawrot, Ulrike Mende, Jin O-Uchi, Gaurav Choudhary
Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1–/–) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1–/– mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1–/– hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1–/– mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction–induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.
Kari T. Chambers, Michael A. Cooper, Alison R. Swearingen, Rita T. Brookheart, George G. Schweitzer, Carla J. Weinheimer, Attila Kovacs, Timothy R. Koves, Deborah M. Muoio, Kyle S. McCommis, Brian N. Finck
Patients with chronic kidney disease (CKD) and end stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, we not only measured the impact of altering the intestinal microbiota on lipid accumulation in coronary arteries, but also examined macrophage lipid uptake and handling pathways in response to uPC. We found atherosclerotic-prone mice fed a high fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, hepatic, and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated uPC exposure increased apoptosis where PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate low density lipoprotein (LDL) uptake and only uPC induced endocytosis and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate uPC itself has a distinct impact on macrophage biology that may contribute to increased cardiovascular risk in patients with CKD.
Lee D. Chaves, Sham Abyad, Amanda M. Honan, Mark A. Bryniarski, Daniel I. McSkimming, Corrine M. Stahura, Steven C. Wells, Donna M. Ruszaj, Marilyn E. Morris, Richard J. Quigg, Rabi Yacoub
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated to functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the non-myocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures of >94% cTnT+ cells. Following purification hiPSC-CMs were re-plated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. Metabolic selected hiPSC-CM monolayers’ phenotype recapitulated many of the functional and structural hallmarks of ischemic cardiomyocytes, including: elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate and abnormal response to β1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generates cell populations with phenotype like what is well known to occur in the setting of ischemic heart failure, and thus provides a novel opportunity for study of human ischemic heart disease.
Justin Davis, Ahmad Chouman, Jeffery Creech, Andre Monteiro da Rocha, Daniela Ponce-Balbuena, Eric N. Jimenez Vazquez, Ruthann Nichols, Andrey Lozhkin, Nageswara R. Madamanchi, Katherine F. Campbell, Todd J. Herron
Left ventricular hypertrophy (LVH) is a primary feature of cardiovascular complications in chronic kidney disease (CKD) patients. MiRNA-30 is an important posttranscriptional regulator of LVH, but it is unknown whether miRNA-30 participates in the process of CKD-induced LVH. In the present study, we found that CKD not only results in LVH but also suppresses miRNA-30 expression in the myocardium. Rescue of cardiomyocyte-specific miRNA-30 attenuates LVH in CKD rats without altering CKD progression. Importantly, in vivo and in vitro knockdown of miRNA-30 in cardiomyocytes leads to cardiomyocyte hypertrophy by upregulating the calcineurin signalling directly. Furthermore, CKD-related detrimental factors, such as fibroblast growth factor-23 (FGF-23), uraemic toxin, angiotensin-II (Ang-II) and transforming growth factor-β (TGF-β), suppress cardiac miRNA-30 expression, while miRNA-30 supplementation blunts cardiomyocyte hypertrophy induced by such factors. These results uncover a novel mechanism of CKD-induced LVH and provide a potential therapeutic target for CKD patients with LVH.
Jingfu Bao, Yinghui Lu, Qin-ying She, Weijuan Dou, Rong Tang, Xiaodong Xu, Mingchao Zhang, Ling Zhu, Qing Zhou, Hui Li, Guohua Zhou, Zhongzhou Yang, Shaolin Shi, Zhihong Liu, Chunxia Zheng
Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu
Though low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we show that proteomic analyses of human and guinea pig heart failure (HF) are consistent a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry reveals 32 and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2mg/kg/day) is sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Though cardiac ATRA declines in both guinea pig HF human IDCM, levels certain retinoid metabolic enzymes diverge. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, abrogates the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.
Ni Yang, Lauren Parker, Jianshi Yu, Jace W. Jones, Ting Liu, Kyriakos N. Papanicolaou, C. Conover Talbot Jr., Kenneth B. Margulies, Brian O'Rourke, Maureen Kane, D. Brian Foster
Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy on preventing heart failure is unknown. Although most DMD patients die from heart failure, cardiomyopathy is undetectable until the teens so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure and dog models show highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevents declines in cardiac function and prohibits onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and non-genetic therapies to optimize cardiac care for DMD patients.
Zachary M. Howard, Lisa E. Dorn, Jeovanna Lowe, Megan D. Gertzen, Pierce C. Ciccone, Neha Rastogi, Guy L. Odom, Federica Accornero, Jeffrey S. Chamberlain, Jill A. Rafael-Fortney
Altered inflammation and tissue remodeling are cardinal features of cardiovascular disease and cardiac transplant rejection. Neutrophils have increasingly been understood to play a critical role in acute rejection and early allograft failure; however, discrete mechanisms that drive this damage remain poorly understood. Herein, we demonstrate that early acute cardiac rejection increases allograft prolyl endopeptidase (PE) in association with de novo production of the neutrophil pro-inflammatory matrikine proline-glycine-proline (PGP). In a heterotopic murine heart transplant model, PGP production and PE activity were associated with early neutrophil allograft invasion and allograft failure. Pharmacologic inhibition of PE with Z-Pro-Prolinal reduced PGP, attenuated early neutrophil graft invasion, and reduced pro-inflammatory cytokine expression. Importantly, these changes helped preserve allograft rejection-free survival and function. Notably, within two independent patient cohorts, both PGP and PE activity were increased among patients with biopsy-proven rejection. The observed induction of PE and matrikine generation provides a novel link between neutrophilic inflammation and cardiovascular injury, represents a potentially new target to reduce allogenic immune responses, and uncovers a previously unrecognized mechanism of cardiovascular disease.
Gregory A. Payne, Nirmal S. Sharma, Charitharth V. Lal, Chunyan Song, Lingling Guo, Camilla Margaroli, Liliana Viera, Siva Kumar, Jindong Li, Melanie Bosley, Dongqi Xing, Xin Xu, J. Michael Wells, James F. George, Jose A. Tallaj, Massoud Leesar, J. Edwin Blalock, Amit Gaggar
A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.
Matthew A. Walker, Juan Chavez, Outi Villet, Xiaoting Tang, Andrew Keller, James E. Bruce, Rong Tian
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