Coupled myovascular expansion directs cardiac growth and regeneration.

Heart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here, we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled. We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth. Our work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.

Therapeutic Targets for Heart Failure Identified Using Proteomics and Mendelian Randomization.

BACKGROUND: Heart failure (HF) is a highly prevalent disorder for which disease mechanisms are incompletely understood. The discovery of disease-associated proteins with causal genetic evidence provides an opportunity to identify new therapeutic targets. METHODS: We investigated the observational and causal associations of 90 cardiovascular proteins, which were measured using affinity-based proteomic assays. First, we estimated the associations of 90 cardiovascular proteins with incident heart failure by means of a fixed-effect meta-analysis of 4 population-based studies, composed of a total of 3019 participants with 732 HF events. The causal effects of HF-associated proteins were then investigated by Mendelian randomization, using cis-protein quantitative loci genetic instruments identified from genomewide association studies in more than 30 000 individuals. To improve the precision of causal estimates, we implemented an Mendelian randomization model that accounted for linkage disequilibrium between instruments and tested the robustness of causal estimates through a multiverse sensitivity analysis that included up to 120 combinations of instrument selection parameters and Mendelian randomization models per protein. The druggability of candidate proteins was surveyed, and mechanism of action and potential on-target side effects were explored with cross-trait Mendelian randomization analysis. RESULTS: Forty-four of ninety proteins were positively associated with risk of incident HF (P<6.0×10-4). Among these, 8 proteins had evidence of a causal association with HF that was robust to multiverse sensitivity analysis: higher CSF-1 (macrophage colony-stimulating factor 1), Gal-3 (galectin-3) and KIM-1 (kidney injury molecule 1) were positively associated with risk of HF, whereas higher ADM (adrenomedullin), CHI3L1 (chitinase-3-like protein 1), CTSL1 (cathepsin L1), FGF-23 (fibroblast growth factor 23), and MMP-12 (matrix metalloproteinase-12) were protective. Therapeutics targeting ADM and Gal-3 are currently under evaluation in clinical trials, and all the remaining proteins were considered druggable, except KIM-1. CONCLUSIONS: We identified 44 circulating proteins that were associated with incident HF, of which 8 showed evidence of a causal relationship and 7 were druggable, including adrenomedullin, which represents a particularly promising drug target. Our approach demonstrates a tractable roadmap for the triangulation of population genomic and proteomic data for the prioritization of therapeutic targets for complex human diseases.

LMNA Cardiomyopathy: Important Considerations for the Heart Failure Clinician.

BACKGROUND: A diagnosis of Lamin proteins A and C cardiomyopathy (LMNA-CM) not only impacts disease prognosis, but also leads to specific guideline-recommended treatment options for these patients. This etiology is fundamentally different from other genetic causes of dilated CM. METHODS AND RESULTS: LMNA-CM often presents early in the third to fourth decades and there is an age-dependent penetrance of nearly 90% among those with a positive genotype for LMNA-CM. Oftentimes, electrical abnormalities with either conduction disturbances and/or either atrial or ventricular arrhythmias manifest before there is imaging evidence of left ventricular dysfunction. Given these subtle early findings, cardiac magnetic resonance provides helpful guidance regarding patterns of enhancement associated with LMNA-CM, often before there is significant left ventricular dilation and/or a decrease in the ejection fraction and could be used for further understanding of risk stratification and prognosis of asymptomatic genotype-positive individuals. Among symptomatic patients with LMNA-CM, approximately one-quarter of individuals progress to needing advanced heart failure therapies such as heart transplantation. CONCLUSIONS: In the era of precision medicine, increased recognition of clinical findings associated with LMNA-CM and increased detection by genetic testing among patients with idiopathic nonischemic CM is of increasing importance. Not only does a diagnosis of LMNA-CM have implications for management and risk stratification, but new gene-based therapies continue to be evaluated for this group. Clinicians must be aware not only of the general indications for genetic testing in arrhythmogenic and dilated cardiomyopathies and of when to suspect LMNA-CM, but also of the clinical trials underway targeted toward the different genetic cardiomyopathies.

Circulating Angiokines Are Associated With Reverse Remodeling and Outcomes in Chronic Heart Failure.

BACKGROUND: We sought to determine whether circulating modifiers of endothelial function are associated with cardiac structure and clinical outcomes in patients with heart failure with reduced ejection fraction (HFrEF). METHODS: We measured 25 proteins related to endothelial function in 99 patients from the GUIDE-IT study. Protein levels were evaluated for association with echocardiographic parameters and the incidence of all-cause death and hospitalization for heart failure (HHF). RESULTS: Higher concentrations of angiopoietin 2 (ANGPT2), vascular endothelial growth factor receptor 1 (VEGFR1) and hepatocyte growth factor (HGF) were significantly associated with worse function and larger ventricular volumes. Over time, decreases in ANGPT2 and, to a lesser extent, VEGFR1 and HGF, were associated with improvements in cardiac size and function. Individuals with higher concentrations of ANGPT2, VEGFR1 or HGF had increased risks for a composite of death and HHF in the following year (HR 2.76 (95% CI 1.73-4.40) per 2-fold change in ANGPT2; HR 1.76 (95% CI 1.11-2.79) for VEGFR1; and HR 4.04 (95% CI 2.19-7.44) for HGF). CONCLUSIONS: Proteins related to endothelial function associate with cardiac size, cardiac function and clinical outcomes in patients with HFrEF. These results support the concept that endothelial function may be an important contributor to the progression to and the recovery from HFrEF.

Cardiac Sarcoidosis: Current Approaches to Diagnosis and Management.

PURPOSE OF REVIEW: Cardiac sarcoidosis (CS) is an important cause of non-ischemic cardiomyopathy and has specific diagnostic and therapeutic considerations. With advances in imaging techniques and treatment approaches, the approach to monitoring disease progression and management of CS continues to evolve. The purpose of this review is to highlight advances in CS diagnosis and treatment and present a center's multidisciplinary approach to CS care. RECENT FINDINGS: In this review, we highlight advances in granuloma biology along with contemporary diagnostic approaches. Moreover, we expand on current targets of immunosuppression focused on granuloma biology and concurrent advances in the cardiovascular care of CS in light of recent guideline recommendations. Here, we review advances in the understanding of the sarcoidosis granuloma along with contemporary diagnostic and therapeutic considerations for CS. Additionally, we highlight knowledge gaps and areas for future research in CS treatment.

Modulation of tissue repair by regeneration enhancer elements.

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for 'tissue regeneration enhancer elements' (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs.

Vegfaa instructs cardiac muscle hyperplasia in adult zebrafish.

During heart development and regeneration, coronary vascularization is tightly coupled with cardiac growth. Although inhibiting vascularization causes defects in the innate regenerative response of zebrafish to heart injury, angiogenic signals are not known to be sufficient for triggering regeneration events. Here, by using a transgenic reporter strain, we found that regulatory sequences of the angiogenic factor vegfaa are active in epicardial cells of uninjured animals, as well as in epicardial and endocardial tissue adjacent to regenerating muscle upon injury. Additionally, we find that induced cardiac overexpression of vegfaa in zebrafish results in overt hyperplastic thickening of the myocardial wall, accompanied by indicators of angiogenesis, epithelial-to-mesenchymal transition, and cardiomyocyte regeneration programs. Unexpectedly, vegfaa overexpression in the context of cardiac injury enabled ectopic cardiomyogenesis but inhibited regeneration at the site of the injury. Our findings identify Vegfa as one of a select few known factors sufficient to activate adult cardiomyogenesis, while also illustrating how instructive factors for heart regeneration require spatiotemporal control for efficacy.

A Roadmap to Heart Regeneration Through Conserved Mechanisms in Zebrafish and Mammals.

PURPOSE OF REVIEW: The replenishment of lost or damaged myocardium has the potential to reverse heart failure, making heart regeneration a goal for cardiovascular medicine. Unlike adult mammals, injury to the zebrafish or neonatal mouse heart induces a robust regenerative program with minimal scarring. Recent insights into the cellular and molecular mechanisms of heart regeneration suggest that the machinery for regeneration is conserved from zebrafish to mammals. Here, we will review conserved mechanisms of heart regeneration and their translational implications. RECENT FINDINGS: Based on studies in zebrafish and neonatal mice, cardiomyocyte proliferation has emerged as a primary strategy for effecting regeneration in the adult mammalian heart. Recent work has revealed pathways for stimulating cardiomyocyte cell cycle reentry; potential developmental barriers for cardiomyocyte proliferation; and the critical role of additional cell types to support heart regeneration. Studies in zebrafish and neonatal mice have established a template for heart regeneration. Continued comparative work has the potential to inform the translation of regenerative biology into therapeutics.

Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.

In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

Myocardial NF-κB activation is essential for zebrafish heart regeneration.

Heart regeneration offers a novel therapeutic strategy for heart failure. Unlike mammals, lower vertebrates such as zebrafish mount a strong regenerative response following cardiac injury. Heart regeneration in zebrafish occurs by cardiomyocyte proliferation and reactivation of a cardiac developmental program, as evidenced by induction of gata4 regulatory sequences in regenerating cardiomyocytes. Although many of the cellular determinants of heart regeneration have been elucidated, how injury triggers a regenerative program through dedifferentiation and epicardial activation is a critical outstanding question. Here, we show that NF-κB signaling is induced in cardiomyocytes following injury. Myocardial inhibition of NF-κB activity blocks heart regeneration with pleiotropic effects, decreasing both cardiomyocyte proliferation and epicardial responses. Activation of gata4 regulatory sequences is also prevented by NF-κB signaling antagonism, suggesting an underlying defect in cardiomyocyte dedifferentiation. Our results implicate NF-κB signaling as a key node between cardiac injury and tissue regeneration.

Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.

Certain lower vertebrates like zebrafish activate proliferation of spared cardiomyocytes after cardiac injury to regenerate lost heart muscle. Here, we used translating ribosome affinity purification to profile translating RNAs in zebrafish cardiomyocytes during heart regeneration. We identified dynamic induction of several Jak1/Stat3 pathway members following trauma, events accompanied by cytokine production. Transgenic Stat3 inhibition in cardiomyocytes restricted injury-induced proliferation and regeneration, but did not reduce cardiogenesis during animal growth. The secreted protein Rln3a was induced in a Stat3-dependent manner by injury, and exogenous Rln3 delivery during Stat3 inhibition stimulated cardiomyocyte proliferation. Our results identify an injury-specific cardiomyocyte program essential for heart regeneration.

Essential and unexpected role of Yin Yang 1 to promote mesodermal cardiac differentiation.

RATIONALE: Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). OBJECTIVE: To identify novel regulators of mesodermal cardiac lineage commitment. METHODS AND RESULTS: We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs. CONCLUSIONS: These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.

Fibronectin is deposited by injury-activated epicardial cells and is necessary for zebrafish heart regeneration.

Unlike adult mammals, adult zebrafish vigorously regenerate lost heart muscle in response to injury. The epicardium, a mesothelial cell layer enveloping the myocardium, is activated to proliferate after cardiac injury and can contribute vascular support cells or provide mitogens to regenerating muscle. Here, we applied proteomics to identify secreted proteins that are associated with heart regeneration. We found that Fibronectin, a main component of the extracellular matrix, is induced and deposited after cardiac damage. In situ hybridization and transgenic reporter analyses indicated that expression of two fibronectin paralogues, fn1 and fn1b, are induced by injury in epicardial cells, while the itgb3 receptor is induced in cardiomyocytes near the injury site. fn1, the more dynamic of these paralogs, is induced chamber-wide within one day of injury before localizing epicardial Fn1 synthesis to the injury site. fn1 loss-of-function mutations disrupted zebrafish heart regeneration, as did induced expression of a dominant-negative Fibronectin cassette, defects that were not attributable to direct inhibition of cardiomyocyte proliferation. These findings reveal a new role for the epicardium in establishing an extracellular environment that supports heart regeneration.

Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5.

RATIONALE: Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. OBJECTIVE: To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). METHODS AND RESULTS: We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. CONCLUSIONS: Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

Incidence and Predictors of Relapse After Weaning Immune Suppressive Therapy in Cardiac Sarcoidosis.

Cardiac sarcoidosis (CS) is a relapsing-remitting disease, and immune suppression (IS) is the mainstay of therapy. Predictors of relapse for patients with CS in remission are not well characterized. We assessed incidence of relapse in consecutive patients with CS treated with high-dose steroids and/or steroid-sparing agents (SSA) in our center from 2000 to 2020. Remission was defined as reaching maintenance therapy (no IS, SSA, and/or prednisone ≤5 mg/d) for ≥1 month. Relapse was defined as recurrence of CS syndrome requiring IS intensification: heart failure, ventricular arrhythmia, decrease in left ventricular ejection fraction, or increased disease burden on imaging. Among 68 patients, the mean age was 50.7±9.0 years; 25 (37%) were women, and 32 (47%) were Black. In total, 59 patients (87%) reached remission. Over a median follow-up of 39.5 months (interquartile range 17.6, 92.5), 28 (48%) relapsed. Greater percentage of late gadolinium enhancement (LGE) on pretreatment magnetic resonance imaging corresponded with increased likelihood of relapse (odds ratio 1.396 per 5% increase [95% confidence interval (CI) 1.04 to 1.88]; p = 0.028). LGE ≥11% predicted elevated risk of relapse (adjusted odds ratio 4.998 [1.34 to 18.64]; p = 0.017). Shorter time to relapse was observed with isolated CS (adjusted hazard ratio 4.084 [1.44,11.56]; p = 0.008) and LGE ≥11% (adjusted hazard ratio 3.007 [1.01, 8.98]; p = 0.049). Approximately 1 in 2 patients with CS in remission experienced relapse. Greater burden of LGE on cardiac magnetic resonance imaging and isolated CS are associated with greater risk of relapse. Future work is needed to refine risk stratification for relapse and to optimize surveillance strategies on the basis of the burden of disease.

An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Recovery of left ventricular function is associated with improved outcomes in LVAD recipients.

BACKGROUND: The significance of recovered left ventricular ejection fraction (LVEF) in LVAD recipients, outside of pump explantation, is unclear. METHODS: Patients undergoing first LVAD implantation at Duke University Hospital between 2006 and 2017 were evaluated for LVEF recovery up to 2 years following implant. Occurrence of gastrointestinal bleeding (GIB), hospitalization for heart failure (HF), pump thrombosis and death were assessed before and after LVEF recovery. RESULTS: Of 286 patients who met inclusion criteria, 9.8% reached a "threshold" of recovery with an LVEF ≥ 40%. 17.4% achieved "relative" recovery with an increase in LVEF ≥ 10% since LVAD implantation. For either definition, recovered patients had a lower incidence of a composite endpoint of GIB, HF hospitalization, pump thrombosis, or death compared to patients without recovery. Patients with "threshold" recovery had 4.7 events per 100 patient-years (95% CI, 0.7-33.6) compared to 48.8 events per 100 patient-years (95% CI, 39.5-60.3) without "threshold" recovery [p = .020]. Those with "relative" recovery had 14.1 events per 100 patient-years [95% CI, 5.9-33.8] versus 50.7 events per 100 patient-years (95% CI, 40.7-63.0) without "relative" recovery [p = 0.005]. However, improved outcomes in the "relative" recovery group were limited to those who also met the "threshold" definition. Importantly, among patients who achieved "threshold" recovery, the incidence of the composite endpoint declines in the postrecovery period, suggesting that LVEF recovery mechanistically results in improved outcomes. CONCLUSIONS: An LVEF ≥ 40% associates with better outcomes in LVAD recipients. Methods to promote recovery could reduce morbidity and mortality related to LVAD support.

Severe chronic low back pain: patient journey from onset of symptoms to strong opioid treatments in Europe.

Aim: We report the first patient roadmap in severe chronic low back pain (cLBP) in Europe, assessing the views of cLBP patients and general practitioners (GPs) who treat cLBP with regard to current cLBP management. Methodology: Patient journey mapping was conducted in four European countries to assess the views of cLBP patients (n = 20) and GPs (n = 40). Results: Four broad phases of cLBP, subdivided into eight individual steps, were identified as part of the patient journey, showing a disconnect between patients' and physicians' treatment goals, and expectations regarding pain relief levels for some patients. Conclusion: Improved communication, with greater involvement of patients in multimodal management decisions, might benefit the GP-patient relationship and overall outcomes for cLBP patients.

Differentiation of Human Induced Pluripotent Stem Cells into Epicardial-Like Cells.

The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle populations of the heart and secrete paracrine factors that promote cardiomyocyte proliferation and angiogenesis. Despite a central role in cardiac biology, the mechanisms by which epicardial cells influence cardiac growth are largely unknown, and robust models of the epicardium are needed. Here, we review our protocol for differentiating induced pluripotent stem cells (iPSCs) into epicardial-like cells through temporal modulation of canonical Wnt signaling. iPSC-derived epicardial cells (iECs) resemble in vivo epicardial cells morphologically and display markers characteristic of the developing epicardium. We also review our protocol for differentiating iECs into fibroblasts and smooth muscle cells through treatment with bFGF and TGF-ß1, respectively. iECs provide a platform for studying fundamental epicardial biology and can inform strategies for therapeutic heart regeneration.