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
Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1) and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death while overexpressing these genes maintained their expansion in hyperoxia, suggesting oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1 and other fatty acid synthesis genes in atrial cardiomyocytes may thus provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.
Ethan David Cohen, Min Yee, George A. Porter, Jr., Erin E. Ritzer, Andrew N. McDavid, Paul S. Brookes, Gloria S. Pryhuber, Michael A. O'Reilly
Myotonic dystrophy type 1 (DM1) is caused by a CTG-repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG-repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-seq results, calcium handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.
Ashish N. Rao, Hannah M. Campbell, Xiangnan Guan, Tarah A. Word, Xander H.T. Wehrens, Zheng Xia, Thomas A. Cooper
Background: Loss-of-function variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including epileptic encephalopathy and cardiac arrhythmia. β1 subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming a subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Methods: We investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase.Results: We show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA-seq, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null cardiac tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. Conclusion: We propose that human disease variants resulting in SCN1B loss-of-function cause transcriptional dysregulation that contributes to altered excitability. These results provide important new insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multi-functionality of sodium channel β1 subunits.
Alexandra A. Bouza, Nnamdi Edokobi, Samantha L. Hodges, Alexa M. Pinsky, James Offord, Lin Piao, Yan-Ting Zhao, Anatoli N. Lopatin, Luis F. Lopez-Santiago, Lori L. Isom
The pathogenesis of preeclampsia and other hypertensive disorders of pregnancy remains poorly-defined despite the substantial burden of maternal and neonatal morbidity associated with these conditions. In particular, the role of genetic variants as determinants of disease susceptibility remains unknown. Storkhead-box protein 1 (STOX1) was first identified as a preeclampsia risk gene through family-based genetic linkage studies in which loss-of-function variants were proposed to underlie increased preeclampsia susceptibility. We generated a genetic Stox1 loss-of-function mouse model (Stox1 KO), to evaluate whether STOX1 regulates blood pressure in pregnancy. Pregnant Stox1 KO mice developed gestational hypertension evidenced by a significant increase in blood pressure compared with wild type by E17.5. While severe renal, placental, or fetal growth abnormalities were not observed, the Stox1 KO phenotype was associated with placental vascular and extracellular matrix abnormalities. Mechanistically, we found that gestational hypertension in Stox1 KO mice resulted from activation of the uteroplacental renin-angiotensin system. We confirmed this mechanism by showing that treatment of pregnant Stox1 KO mice with an angiotensin II receptor blocker rescued the phenotype. Our study demonstrates the utility of genetic mouse models for uncovering links between genetic variants and effector pathways implicated in the pathogenesis of hypertensive disorders of pregnancy.
Jacqueline G. Parchem, Keizo Kanasaki, Soo Bong Lee, Megumi Kanasaki, Joyce L. Yang, Yong Xu, Kadeshia M. Earl, Rachel A. Keuls, Vincent H. Gattone, Raghu Kalluri
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