Maladaptive Cardiac Remodeling Research Articles

RKER-012, a novel modified ActRIIB ligand trap, attenuated right ventricular cardiomyopathy in a preclinical model of pulmonary arterial hypertension

Abstract Background The primary cause of death in pulmonary arterial hypertension (PAH) is right ventricular (RV) failure. Initially, the RV adapts to heightened pressure through adaptive remodeling, but prolonged pressure overload triggers maladaptive remodeling, culminating in failure. Overactive activin/growth differentiation factor (GDF) signaling has been implicated in cardiomyopathy and heart failure. RKER-012, a research version of KER-012, is an investigational modified activin receptor type IIB (ActRIIB) ligand trap designed to specifically bind and inhibit select TGF-β ligands, including activins A and B and GDFs 8 and 11. RKER-012 previously exhibited a cardioprotective effect in a pulmonary arterial banding (PAB) model of RV dysfunction. To determine the effect of RKER-012 on afterload-induced RV cardiomyopathy caused by increased pulmonary artery pressure, we utilized an angio-obliterative Sugen/hypoxia (SH) rat model of PAH that closely mimics human PAH pathology. Methods Male Sprague Dawley rats were subcutaneously (s.c.) injected with one dose of Sugen 5416 and exposed to 10% hypoxia for 3 weeks during which they received either vehicle (SH-Veh; s.c; BIW) or RKER-012 (10 mg/kg; s.c; BIW). Normoxic (Nx) control rats were maintained in room air for 3 weeks with BIW s.c. vehicle treatment. After 3 weeks of treatment, systolic pulmonary arterial pressure (sPAP) and Fulton index (FI) were assessed. Hallmark genes of inflammatory process, endothelial/platelet activation, and fibrosis were evaluated in the RV by qPCR. Results Increases in FI (+99.4%;p<0.0001) and sPAP (+250.9%;p<0.0001) were observed in SH-Veh rats vs. Nx rats, consistent with the development of pulmonary and cardiac impairment. In contrast, treatment with RKER-012 reduced FI (-28.0%;p<0.001) and sPAP (-44.5%;p<0.001). In the RV, SH-Veh rats had increased expression of TGF-ß pathway genes involved in inflammation, endothelial/platelet activation, and fibrosis. RKER-012 treatment reduced expression of these genes (Tgf-β1, -44.7%;p=0.003; Fstl3, -43.5%;p=0.0162; Mcp1, -22.5%;p=0.535; Cd68, -60.3%;p=0.0048; Ctgf, -57.1%;p=0.0139; Col1a1, -47.2%, p=0.0255 ;Col3a1, -69.5%;p<0.0001; Lox, -61.4%;p=0.0054; P-selectin, -63.9%;p=0.018) in comparison to SH-Veh treatment. Conclusion Consistent with previous findings in a PAB mouse model, RKER-012 treatment protected against increased sPAP and maladaptive cardiac remodeling in a SH rat model of PAH. Additionally, RKER-012 attenuated RV cardiomyopathy by reducing the expression of genes involved in inflammatory and fibrotic processes. These preclinical findings in the RV paralleled the changes in circulating cardiovascular health biomarkers, including reduced NT-proBNP, observed in a Phase 1 trial of KER-012 in healthy volunteers. These findings, along with the safety and tolerability observed in the Phase 1 trial, provided rationale for the ongoing Phase 2 TROPOS trial of KER-012 in patients with PAH (NCT05975905).

The Extracellular Matrix and Cardiac Pressure Overload: Focus on Novel Treatment Targets.

Heart failure is a significant health issue in developed countries, often stemming from conditions like hypertension, which imposes a pressure overload on the heart. Despite various treatment strategies for heart failure, many lack long-term effectiveness. A critical aspect of cardiac disease is the remodeling of the heart, where compensatory changes in the extracellular matrix exacerbate disease progression. This review explores the processes and changes occurring in the pressure-overloaded heart with respect to the extracellular matrix. It further summarizes current treatment strategies, and then focuses on novel treatment targets for maladaptive cardiac remodeling, derived from transverse aortic constriction-induced pressure overload animal models.

Mesenchymal stem cells arouse myocardial NAD+ metabolism to alleviate microgravity-induced cardiac dysfunction

After prolonged space operations, astronauts showed maladaptive atrophy within mostly left-ventricular myocardium, resulting in cardiac dysfunction. However, the mechanism of cardiac dysfunction under microgravity conditions is unclear, and the relevant prevention and treatment measures also need to be explored. Through simulating the microgravity environment with a tail suspension (TS) model, we found that long-term exposure to microgravity promotes aging of mouse hearts, which is closely related to cardiac dysfunction. The intravenous administration of adipose-derived mesenchymal stem cells (ADSCs) emerged preventive and therapeutic effect against myocardial senescence and the decline in cardiac function. Plasma metabolomics analysis suggests the loss of NAD+ in TS mice and motivated myocardial NAD + metabolism and utilization in ADSCs-treated mice, likely accounting for ADSCs' function. Oral administration of nicotinamide mononucleotide (NMN, a NAD + precursor) showed similar therapeutic effect to ADSCs treatment. Collectively, these data implicate the effect of ADSCs in microgravity-induced cardiac dysfunction and provide new therapeutic ideas for aging-related maladaptive cardiac remodeling.

CITED4 gene therapy protects against maladaptive cardiac remodeling after ischemia/reperfusion injury in mice

Cardiac signaling pathways functionally important in the heart's response to exercise often protect the heart against pathological stress, potentially providing novel therapeutic targets. However, it is important to determine which of these pathways can be feasibly targeted invivo. Transgenic overexpression of exercise-induced CITED4 has been shown to protect against adverse remodeling after ischemia/reperfusion injury (IRI). Here we investigated whether somatic gene transfer of CITED4 in a clinically relevant time frame could promote recovery after IRI. Cardiac CITED4 gene delivery via intravenous AAV9 injections in wild type mice led to an approximately 3-fold increase in cardiac CITED4 expression. After 4weeks, CITED4-treated animals developed physiological cardiac hypertrophy without adverse remodeling. In IRI, delivery of AAV9-CITED4 after reperfusion resulted in a 6-fold increase in CITED4 expression 1week after surgery, as well as decreased apoptosis, fibrosis, and inflammatory markers, culminating in a smaller scar and improved cardiac function 8weeks after IRI, compared with control mice receiving AAV9-GFP. Somatic gene transfer of CITED4 induced a phenotype suggestive of physiological cardiac growth and mitigated adverse remodeling after ischemic injury. These studies support the feasibility of CITED4 gene therapy delivered in a clinically relevant time frame to mitigate adverse ventricular remodeling after ischemic injury.

Fenofibrate reduces cardiac remodeling by mitochondrial dynamics preservation in a renovascular model of cardiac hypertrophy

Fenofibrate, a PPAR-α agonist clinically used to lower serum lipid levels, reduces cardiac remodeling and improves cardiac function. However, its mechanism of action is not completely elucidated. In this study we examined the effect of fenofibrate on mitochondria in a rat model of renovascular hypertension, focusing on mediators controlling mitochondrial dynamics and autophagy.Rats with two-kidney one-clip (2K1C) hypertension were treated with fenofibrate 150 mg/kg/day (2K1C-FFB) or vehicle (2K1C-VEH) for 8 weeks. Systolic blood pressure and cardiac functional were in-vivo assessed, while cardiomyocyte size and protein expression of mediators of cardiac hypertrophy and mitochondrial dynamics were ex-vivo examined by histological and Western blot analyses.Fenofibrate treatment counteracted the development of hypertension and the increase of left ventricular mass, relative wall thickness and cross-sectional area of cardiomyocytes. Furthermore, fenofibrate re-balanced the expression Mfn2, Drp1 and Parkin, regulators of fusion, fission, mitophagy respectively. Regarding autophagy, the LC3-II/LC3-I ratio was increased in 2K1C-VEH and 2K1C-FFB, whereas the autophagy was increased only in 2K1C-FFB.In cultured H9C2 cardiomyoblasts, fenofibrate reversed the Ang II-induced mRNA up-regulation of hypertrophy markers Nppa and Myh7, accumulation of reactive oxygen species and depolarization of the mitochondrial membrane exerting protection mediated by up-regulation of the Uncoupling protein 2.Our results indicate that fenofibrate acts directly on cardiomyocytes and counteracts the pressure overload-induced cardiac maladaptive remodeling. This study reveals a so far hidden mechanism involving mitochondrial dynamics in the beneficial effects of fenofibrate, support its repurposing for the treatment of cardiac hypertrophy and provide new potential targets for its pharmacological function.

Therapeutic Inhibition of LincRNA-p21 Protects Against Cardiac Hypertrophy.

Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 (nuclear factor of activated T cells/myocyte enhancer factor-2) pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1 (KRAB-associated protein-1). lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

Moderate aortic stenosis: Navigating the uncharted.

Aortic stenosis (AS) stands as the most common valvular heart disease in developed countries and is characterized by progressive narrowing of the aortic valve orifice resulting in elevated transvalvular flow resistance, left ventricular hypertrophy, and progressive increased risk of heart failure and sudden death. This narrative review explores clinical challenges and evolving perspectives in moderate AS, where discrepancies between aortic valve area and pressure gradient measurements may pose diagnostic and therapeutic quandaries. Transthoracic echocardiography is the first-line imaging modality for AS evaluation, yet cases of discordance may require the application of ancillary noninvasive diagnostic modalities. This review underscores the importance of accurate grading of AS severity, especially in low-gradient phenotypes, emphasizing the need for vigilant follow-up. Current clinical guidelines primarily recommend aortic valve replacement for severe AS, potentially overlooking latent risks in moderate disease stages. The noninvasive multimodality imaging approach-including echocardiography, cardiac magnetic resonance, computed tomography, and nuclear techniques-provides unique insights into adaptive and maladaptive cardiac remodeling in AS and offers a promising avenue to deliver precise indications and exact timing for intervention in moderate AS phenotypes and asymptomatic patients, potentially improving long-term outcomes. Nevertheless, what we may have gleaned from a large amount of observational data is still insufficient to build a robust framework for clinical decision-making in moderate AS. Future research will prioritize randomized clinical trials designed to weigh the benefits and risks of preemptive aortic valve replacement in the management of moderate AS, as directed by specific imaging and nonimaging biomarkers.

Cardiac sympathetic overdrive, M2 macrophage activation and fibroblast heterogeneity are associated with cardiac remodeling in a chronic pressure overload rat model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is a multifaceted pathogenesis disease and the exact mechanisms driving HFpEF have not been completely elucidated. Pressure overload hypertrophy (POH) related fibroblasts and M2 macrophages in HFpEF myocardium have been recently identified and are now of great interest. Sympathetic overdrive has also been implicated in HFpEF. This study is designed to dynamically observe the potential roles of aforementioned mechanisms in pathological remodeling and cardiac dysfunction in chronic PO rats. Surgical constriction of the abdominal aorta was used for induction of HFpEF. Echocardiography, electrocardiogram, hemodynamic measurement, hematoxylin and eosin staining, Masson staining, immunohistochemistry and immunofluorescence were performed to assess the changes in heart dysfunction, cardiac remodeling and driving mechanisms at different time points (2, 18, 24weeks). The PO induced HFpEF model was well established, which was confirmed by the persistent increase in carotid artery systolic and diastolic blood pressure, and left ventricle hypertrophy at the corresponding postoperative stage. Meanwhile, PO hypertrophy gradually developed into HFpEF, associated with QT and QTc intervals prolongation, normal systolic (EF was maintained at >50%) but impaired diastolic function (increasing LVEDP and LV -dP/dtmin, abnormal E/A ratio), increased myocytes size, and observed relatively slight inflammatory infiltration but robust reactive fibrosis. IHC staining further confirmed that macrophages (CD68) but not neutrophils (MPO) or T cells (CD3) accounted for a predominant proportion of infiltrating cells. Mechanistically, we found that the infiltrating macrophages in the heart expressed high levels of CD206 which was simultaneously adjacent to POH fibroblasts appeared to overexpression of α-SMA in PO rats at late stages. Interestingly, we distinguished two different POHF sub-populations during PO induced HFpEF development, according to non overlapping signals of α-SMA and PDGFRα/β proteins. Additionally, PO led to a pronounced exaggeration in sympathetic fibers at all time points. These findings suggest that the establishing model here begins with cardiac sympathetic overdrive, subsequently along with immune cells especially M2 macrophage accumulation and fibroblast heterogeneity at later stages is associated with the development of cardiac maladaptive remodeling and diastolic dysfunction thus further progression to HFpEF.

Early Initiation of SGLT2 Inhibitor is Important Irrespective of Ejection Fraction

Introduction Sodium Glucose Cotransporter inhibitor (SGLTi) has revolutionized the medical management of heart failure in the last decade. This molecule was initially described as a glucose-lowering agent but now it has been recommended in patients with chronic heart failure irrespective of ejection fraction and diabetic status. SGLT2 inhibitor reduces preload by natriuresis and diuresis, reduces afterload, decreases pulmonary artery pressure, improves myocardial energetics, and decreases myocardial fibrosis. It also reduces NTpro BNP levels in heart failure patients, decreases serum uric acid, and promotes weight loss. It has also been proposed that this molecule has a favorable effect on vascular remodeling. SGLT-2i improves cardiac output and attenuates maladaptive cardiac remodeling, chronic inflammation, oxidative stress, and endothelial dysfunction (ED) by restoring the activity of nitric oxide (NO) within the vascular endothelium. As was foreseeable this molecule became one of the foundation pillars in the management of heart failure with reduced as well as preserved ejection fraction. SGLT2i also has a reno-protective effect, particularly in patients with heart failure and diabetes. Evidence with SGLT2 inhibitor in Heart failure with preserved ejection fraction (HFpEF) DELIVER TRIAL has evaluated the effect of dapagliflozin on total (first and recurrent) heart failure events in patients with heart failure having preserved ejection fraction (HFpEF) or mildly reduced ejection fraction (HFmrEF). A total of 6263 patients with LVEF of more than 40 %, NHYA Class II-IV, and high NT-pro BNP (>300pg/ml in sinus rhythm and > 600pg/ml in patients with atrial fibrillation) were included over the total study period of two years. Dapagliflozin 10 mg was compared to placebo. In patients with HFmrEF and HFpEF, dapagliflozin reduced the risk of total heart failure events and heart failurerelated hospital admissions. (1)

Role of Perilipins in Oxidative Stress-Implications for Cardiovascular Disease.

Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and antioxidants in a cell. In the heart, oxidative stress may deteriorate calcium handling, cause arrhythmia, and enhance maladaptive cardiac remodeling by the induction of hypertrophic and apoptotic signaling pathways. Consequently, dysregulated ROS production and oxidative stress have been implicated in numerous cardiac diseases, including heart failure, cardiac ischemia-reperfusion injury, cardiac hypertrophy, and diabetic cardiomyopathy. Lipid droplets (LDs) are conserved intracellular organelles that enable the safe and stable storage of neutral lipids within the cytosol. LDs are coated with proteins, perilipins (Plins) being one of the most abundant. In this review, we will discuss the interplay between oxidative stress and Plins. Indeed, LDs and Plins are increasingly being recognized for playing a critical role beyond energy metabolism and lipid handling. Numerous reports suggest that an essential purpose of LD biogenesis is to alleviate cellular stress, such as oxidative stress. Given the yet unmet suitability of ROS as targets for the intervention of cardiovascular disease, the endogenous antioxidant capacity of Plins may be beneficial.

Bioenergetic Metabolism Modulatory Peptide Hydrogel for Cardiac Protection and Repair After Myocardial Infarction

AbstractMyocardial infarction (MI) remains a major threat to human health due to the limited energy supply, disordered cell metabolism, massive cardiomyocyte death, and restricted regeneration. Although currently available therapies may relieve myocardial damage, restoring the dysregulated energy metabolism to normal levels has not yet been achieved. MOTS‐c has recently been identified as a regulator of biological metabolism to combat aging; however, its role in reprogramming cardiac metabolism remains to be elucidated. Here, MOTS‐c is chemically conjugated to self‐assembling Q11 peptide to fabricate an injectable hydrogel (MQgel) aimed to improve mitochondria function and cardiomyocyte metabolism post‐MI. It is observed that MQgel effectively protects mitochondria from oxidative damage and normalized cardiomyocyte metabolism, including glucose uptake, glycolysis, and the tricarboxylic acid (TCA) cycle, thereby inhibiting cardiomyocyte death and enhancing cardiomyocyte activity. In a rat MI model, intramyocardial injection of MQgel successfully minimizes the infarct area and fibrosis, promotes angiogenesis, suppresses myocardial hypertrophy, and improves cardiomyocyte survival and metabolic enzyme activity, all of which collaboratively attenuate the maladaptive cardiac remodeling and boost cardiac function and tissue repair. The findings suggest that the self‐assembled mitochondria metabolism‐regulatory peptide hydrogel effectively treats MI, and cellular bioenergy modulation provides a new therapeutic approach for tissue repair after injury.

DNA methylation profiling of circulating CD4+ T cells and network analysis reveal that JUNB, SETD7, and MEF2D genes predict patients with heart failure preserved vs. reduced ejection fraction

Abstract Background Heart failure (HF) with preserved ejection fraction (HFpEF) represents about 50% of new HF diagnosis but still lacks specific biomarkers and effective therapy. Epigenetic-sensitive changes accumulated over time, mainly guided by DNA methylation and demethylation, can lead to chromatin remodelling and affect transcriptional molecular networks underlying microvascular inflammation, oxidative stress, and maladaptive cardiac remodelling which predispose synergistically to HFpEF. Purpose We hypothesize that circulating genome-wide DNA methylation profiles may identify novel biomarkers for discriminating HFpEF vs. HF with reduced EF (HFrEF) and provide insight into the underlying pathophysiology of disease. Methods A total of n=47 subjects were enrolled including patients with HFpEF (n=22) and HFrEF (n=13) as well as healthy subjects (n=12) serving as control group (CON). Starting from pheripheral blood biospecimens, we isolated CD4+ T cells and extracted genomic DNA for genome-wide methylome analysis and total RNA for experimental validation set. Results The reduced representation bisulfite sequencing (RRBS) revealed that patients with HFpEF had unique variations in DNA methylation profiles as compared to HFrEF and CON groups and reflected distinct pathogenic mechanisms. Using the list of differentially methylated CpG sites in HFpEF, we performed a complex network analysis to indentify the "HFpEF interactome" (Fig.1 A-B). Then, using the DisGeNET database we extracted n=12 candidate genes which were already associated with diastolic dysfunction, cardiac hypertrophy, cardiac fibrosis, inflammation, obesity, and hypertension, as key pathophenotypes of HFpEF (Fig.1 C). The hub genes included the ACTB, VAV2, JUNB, HOOK2, SETD7, PNKP, ASAP1, PAICS, DNM1L, HIF1AN, MEF2D, and NRG1. Validation experiment set by qRT-PCR revealed that elevated mRNA levels of the JUNB, SETD7, and MEF2D hypermethylated genes significantly discriminated HFpEF patients vs. CON (p<0.01, p<0.01, p<0.001, respectively) and vs. HFrEF patients (p<0.05, p<0.01, p<0.001, respectively) (Fig. 2A). Receiver operating characteristic (ROC) curve showed that network-oriented JUNB (AUC: 1, p<0.001), SETD7 (AUC: 0.97, p<0.01), and MEF2D (AUC: 1, p<0.001) genes have a high diagnostic accuracy in discriminating HFpEF vs. CON as well as HFpEF vs. HFrEF (AUC: 1, p<0.01, AUC: 0.97, p<0.01, AUC: 1, p<0.001, respectively) (Fig. 2B). Conclusions Circulating CD4+ T cell-derived hypermethylation of JUNB, SETD7, and MEF2D may reveal novel molecular drivers of HFpEF and suggest possible biomarkers to optimize patient phenotyping.

Regulatory roles of the N6-methyladenosine demethylase FTO in cardiac remodeling

Abstract Background The most prevalent internal RNA modification, N6-methyladenosine (m6A), serves as an additional layer of transcriptional and translational regulation. Methyl transferases (like METTL3) and demethylases (e.g., FTO) are the enzymes that catalyze m6A methylations. Our result showed that loss of FTO causes maladaptive cardiac remodeling in rodent hearts, which is consistent with other studies that emphasized the importance of FTO-dependent m6A RNA methylation in maintaining cardiac homeostasis, ventricular hypertrophy, and heart failure. Despite the fact that FTO is essential for stress-induced cardiac hypertrophy, its underlying functional mechanisms in regulating maladaptive cardiac remodeling are not yet known. Purpose In this study, we showed the necessity of FTO dependent m6A methylation in coordinating the molecular and signaling events that are engaged in cardiac hypertrophy, and further identified the detrimental signaling mechanisms that are regulated by FTO during pathological remodeling. Methods Firstly, the control and FTO-cKO (cardiomyocyte specific FTO knockout) group mice were subjected to sham and TAC (transverse aortic constriction) operation. Echocardiography was performed 1 week post-TAC. Secondly, m6A RNA immunoprecipitation (meRIP) sequencing were performed from the isolated mice heart tissue. Additionally, the effect of apoptosis and mTORC1 upon FTO depletion were analysed in both rodents and iPS-CMs. In vitro the interrelation between FTO demethylase, mTORC1 and autophagy in the regulation of cardiac apoptosis were identified by 1. inhibiting mTORC1 pathway with specific inhibitors, and by 2. inhibiting 'late autophagy' in FTO silenced iPS-CMs. Results In contrast to control mice, which exhibit concentric hypertrophy at the beginning of induced pressure overload (PO), echo analysis of one week post-TAC revealed that FTOcKO mice develop a DCM-like phenotype. Further, MeRIP-seq data showed that there is deregulation in the m6A methylation with FTO depletion. Around 114 differently hypermethylated transcripts and 58 differentially hypomethylated transcripts were discovered in FTOcKO sham as compared to Control sham; these transcripts are involved in the control of apoptosis, the mTORC1 signaling pathway, lysosomal biogenesis, and autophagy. In both rodents and iPS-CMs, FTO ablation dramatically enhances cardiac apoptosis and the mTORC1 signaling pathway. Furthermore, inhibition of mTORC1 pathway attenuates cardiac apoptosis in siFTO-iPS-CMs. Moreover, autophagic flux analysis revealed that FTO depletion cause autophagic flux impairment and exacerbate cardiac apoptosis. However, the autophagic flux is ameliorated with mTORC1 inhibition in late-autophagy inhibited siFTO-iPS-CMs. Conclusion Our data unravel the novel roles of FTO demethylase in the course of cardiac remodeling, which opens doors to therapeutic interventions in the treatment of cardiac hypertrophy and heart failure.

Palmitoylation-dependent regulation of cardiomyocyte Rac1 signaling activity and minor effects on cardiac hypertrophy

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling.

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

Abstract P3042: Cardiomyocyte MicroRNA-125a-5p Plays A Vital Protective Role In Myocardial Infarction And Directly Represses Proapoptotic GTP-Cyclohydrolase I Feedback Regulator

Background: We reported that microRNA-125a-5p (miR-125a) is activated by β-blocker carvedilol acting through β-arrestin1-mediated β1-adrenergic receptor (receptor found mainly in cardiomyocytes [CMs]) signaling (Figure A). GTP cyclohydrolase I feedback regulator (GCHFR) inhibits tetrahydrobiopterin which is protective in myocardial infarction (MI). Despite these findings, the roles of miR-125a and GCHFR in MI and their mechanisms of actions remain elusive. Here, we hypothesize that miR-125a in CMs plays a protective role in MI in part by repressing GCHFR, a novel target of miR-125a. Methods and Results: Fractionation of cardiac cell types isolated from mouse hearts reveals that miR-125a expression is higher in CMs than other cells. Using cultured CM and in vivo approaches, we show that miR-125a is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125a exhibit an increased sensitivity to apoptosis, whereas CMs overexpressing miR-125a have increased phospho-AKT prosurvival signaling. MiR-125a is downregulated in failing hearts, and miR-125a overexpression protects mouse hearts against MI. Systemic deletion of miR-125a in mice worsens maladaptive post-MI remodeling. Genome-wide profiling in miR-125a KO mouse hearts identifies GCHFR as a novel target of miR-125a. Mechanistically, protective roles of miR-125a in CMs are in part attributed to the direct and functional repression of proapoptotic GCHFR. Last, novel conditional CM-specific miR-125a KO in mice exacerbates maladaptive cardiac remodeling after MI (Figure B). Conclusions: Our findings suggest a crucial role for the miR-125a/GCHFR axis in regulating CM survival post-MI.

Abstract P2030: Regulation Of Pathologic Cardiac Signaling Via Rac1 Palmitoylation

Development of more effective therapies to prevent maladaptive cardiac remodeling and heart failure would be greatly aided by the identification of novel signaling pathways that regulate cardiac hypertrophy. Cysteine palmitoylation is a reversible post-translational lipid modification that dynamically regulates intracellular trafficking and subcellular compartmentalization of signaling proteins. Previous work from our group identified that mice with cardiomyocyte (CM)-specific overexpression of zDHHC3, a Golgi-localized S-acyltransferase, develop lethal dilated cardiomyopathy. S-acyl-proteomics revealed Cys-178 of Rac1, a small GTPase, as a palmitoylation site modified by zDHHC3 and hearts from zDHHC3-overexpressing mice display elevated levels of palmitoylated, active, and total Rac1, suggesting Rac1 palmitoylation promotes its activation and may drive pathologic cardiac remodeling. Rac1-dependent activation of NADPH oxidase 2 (Nox2) in CMs is required for angiotensin II (AngII)-induced cardiac hypertrophy and myocardial oxidative stress, suggesting inhibition of Rac1/Nox2 signaling could potentially ameliorate adverse cardiac remodeling. We found Rac1 palmitoylation at Cys-178 to be required for hypertrophy and oxidative stress in neonatal rat CMs as reactive oxygen species production and hypertrophy elicited by expression of a constitutively active Rac1 mutant were attenuated by mutation of the Cys-178 palmitoylation site, suggesting palmitoylation targets Rac1 to the Nox2 complex to promote myocardial oxidative stress. Notably, Rac1 palmitoylation and oxidative stress were also enhanced in hearts of mice subjected to AngII infusion and transgenic mice with CM-specific overexpression of the angiotensin II receptor type I (AT1R), suggesting a role for Rac1 palmitoylation in AngII-induced maladaptive cardiac signaling. To further elucidate the role of Rac1 palmitoylation in cardiac pathology, we developed a CM-specific knock-in mouse model in which Cys-178 of Rac1 is mutated to serine. Ongoing work utilizing this model will investigate mechanisms by which palmitoylated and depalmitoylated Rac1 differentially regulate signaling, oxidative stress, and hypertrophy in CMs in response to pathologic stimuli.

T-Cell MyD88 Is a Novel Regulator of Cardiac Fibrosis Through Modulation of T-Cell Activation.

Cardiac inflammation in heart failure is characterized by the presence of damage-associated molecular patterns, myeloid cells, and T cells. Cardiac damage-associated molecular patterns provide continuous proinflammatory signals to myeloid cells through TLRs (toll-like receptors) that converge onto the adaptor protein MyD88 (myeloid differentiation response 88). These induce activation into efficient antigen-presenting cells that activate T cells through their TCR (T-cell receptor). T-cell activation results in cardiotropism, cardiac fibroblast transformation, and maladaptive cardiac remodeling. T cells rely on TCR signaling for effector function and survival, and while they express MyD88 and damage-associated molecular pattern receptors, their role in T-cell activation and cardiac inflammation is unknown. We performed transverse aortic constriction in mice lacking MyD88 in T cells and analyzed remodeling, systolic function, survival, and T-cell activation. We profiled wild type versus Myd88-/- mouse T cells at the transcript and protein level and performed several functional assays. Analysis of single-cell RNA-sequencing data sets revealed that MyD88 is expressed in mouse and human cardiac T cells. MyD88 deletion in T cells resulted in increased levels of cardiac T-cell infiltration and fibrosis in response to transverse aortic constriction. We discovered that TCR-activated Myd88-/- T cells had increased proinflammatory signaling at the transcript and protein level compared with wild type, resulting in increased T-cell effector functions such as adhesion, migration across endothelial cells, and activation of cardiac fibroblast. Mechanistically, we found that MyD88 modulates T-cell activation and survival through TCR-dependent rather than TLR-dependent signaling. Our results outline a novel intrinsic role for MyD88 in limiting T-cell activation that is central to tune down cardiac inflammation during cardiac adaptation to stress.

SPRR1A is a key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation

MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (Sprr1a) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Here, using a novel miR-150 knockout;Sprr1a-hypomorphic (Sprr1ahypo/hypo) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. Moreover, HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

20(S)-ginsenoside Rg3 exerts anti-fibrotic effect after myocardial infarction by alleviation of fibroblasts proliferation and collagen deposition through TGFBR1 signaling pathways

BackgroundMyocardial fibrosis post-myocardial infarction (MI) can induce maladaptive cardiac remodeling as well as heart failure. Although 20(S)-ginsenoside Rg3 (Rg3) has been applied to cardiovascular diseases, its efficacy and specific molecular mechanism in myocardial fibrosis are largely unknown. Herein, we aimed to explore whether TGFBR1 signaling was involved in Rg3's anti-fibrotic effect post-MI. MethodsLeft anterior descending (LAD) coronary artery ligation-induced MI mice and TGF-β1-stimulated primary cardiac fibroblasts (CFs) were adopted. Echocardiography, hematoxlin-eosin and Masson staining, Western-blot and immunohistochemistry, CCK8 and Edu were used to study the effects of Rg3 on myocardial fibrosis and TGFBR1 signaling. The combination mechanism of Rg3 and TGFBR1 was explored by surface plasmon resonance imaging (SPRi). Moreover, myocardial Tgfbr1-deficient mice and TGFBR1 adenovirus were adopted to confirm the pharmacological mechanism of Rg3. ResultsIn vivo experiments, Rg3 ameliorated myocardial fibrosis and hypertrophy and enhanced cardiac function. Rg3-TGFBR1 had the 1.78 × 10−7 M equilibrium dissociation constant based on SPRi analysis, and Rg3 inhibited the activation of TGFBR1/Smads signaling dose-dependently. Cardiac-specific Tgfbr1 knockdown abolished Rg3's protection against myocardial fibrosis post-MI. In addition, Rg3 down-regulated the TGF-β1-mediated CFs growth together with collagen production in vitro through TGFBR1 signaling. Moreover, TGFBR1 adenovirus partially blocked the inhibitory effect of Rg3. ConclusionRg3 improves myocardial fibrosis and cardiac function through suppressing CFs proliferation along with collagen deposition by inactivation of TGFBR1 pathway.