Transverse Aortic Constriction Mice Research Articles

Loss of ADAM15 in female mice does not worsen pressure overload cardiomyopathy, independent of ovarian hormones.

Cardiac hypertrophy is a common feature in several cardiomyopathies. We previously reported that loss of ADAM15 (disintegrin and metalloproteinase 15) worsened cardiac hypertrophy and dilated cardiomyopathy following cardiac pressure overload. Here, we investigated the impact of ADAM15 loss in female mice following cardiac pressure overload induced by transverse aortic constriction (TAC). Female Adam15-/-mice developed the same degree of cardiac hypertrophy, dilation and dysfunction as the parallel female wildtype (WT) mice at 6 weeks post-TAC. To determine if this is due to the protective effects of estrogen which could mask the negative impact of Adam15 loss, WT and Adam15-/- mice underwent ovariectomy (OVx) 2 weeks prior to TAC. Cardiac structure and function analyses were performed at 6 weeks post-TAC. OVx similarly impacted females of both genotypes post-TAC. Calcineurin (Cn) activity was increased post-OVx-TAC, and more in Adam15-/- mice, however this increase was not reflected in the total-to-phospho NFAT levels. Integrin α7 expression, which was upstream of Cn activation in male Adam15-/--TAC mice, remained unchanged in female mice. However, activation of the Mitogen Activated Protein Kinases (ERK, JNK, P38) were greater in Adam15-/--OVx-TAC compared to WT-OVx-TAC mice. In addition, ADAM15 protein levels were significantly increased post-TAC in male but not in female WT mice. Myocardial fibrosis was comparable in non-OVx WT-TAC and Adam15-/--TAC mice. OVx increased the perivascular fibrosis more in Adam15-/- compared to WT mice post-TAC. Our data demonstrate that loss of ovarian hormones did not fully replicate the male phenotype in the female Adam15-/- mice post-TAC. Since ADAM15 levels were increased in males but not in females post-TAC, it is plausible that ADAM15 does not play a prominent role in post-TAC events in female mice. Our findings highlight the significance of factors other than sex hormones in mediating cardiomyopathies in females, which require a more thorough understanding.

The functional role of circRNA CHRC through miR-431-5p/KLF15 signaling axis in the progression of heart failure

Pathological myocardial hypertrophy is a common early clinical manifestation of heart failure, with noncoding RNAs exerting regulatory influence. However, the molecular function of circular RNAs (circRNAs) in the progression from cardiac hypertrophy to heart failure remains unclear. To uncover functional circRNAs and identify the core circRNA signaling pathway in heart failure, we construct a global triple network (microRNA, circRNA, and mRNA) based on the competitive endogenous RNA (ceRNA) theory. We observe that cardiac hypertrophy related circRNA (circRNA CHRC), within the ceRNA network, is down-regulated in both transverse aortic constriction (TAC) mice and Ang-II--treated primary mouse cardiomyocytes. Silencing circRNA CHRC increases cross-sectional cell area, atrial natriuretic peptide, and β-myosin heavy chain levels in primary mouse cardiomyocytes. Further screening reveals that circRNA CHRC targets the miR-431-5p/KLF15 axis implicated in heart failure progression in vivo and in vitro. Immunoprecipitation with anti-Ago2-RNA confirms the interaction between circRNA CHRC and miR-431-5p, while miR-431-5p mimics reverse Klf15 activation caused by circRNA CHRC overexpression. In summary, circRNA CHRC attenuates cardiac hypertrophy via sponging miR-431-5p to maintain the normal level of Klf15 expression.

Treatment with αvβ3-integrin-specific 29P attenuates pressure-overload induced cardiac remodelling after transverse aortic constriction in mice

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvβ3-specific RGD-mimetic, 29P, was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 μg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvβ3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Ketones provide an extra source of fuel for the failing heart without impairing glucose oxidation

BackgroundCardiac glucose oxidation is decreased in heart failure with reduced ejection fraction (HFrEF), contributing to a decrease in myocardial ATP production. In contrast, circulating ketones and cardiac ketone oxidation are increased in HFrEF. Since ketones compete with glucose as a fuel source, we aimed to determine whether increasing ketone concentration both chronically with the SGLT2 inhibitor, dapagliflozin, or acutely in the perfusate has detrimental effects on cardiac glucose oxidation in HFrEF, and what effect this has on cardiac ATP production. Methods8-week-old male C57BL6/N mice underwent sham or transverse aortic constriction (TAC) surgery to induce HFrEF over 3 weeks, after which TAC mice were randomized to treatment with either vehicle or the SGLT2 inhibitor, dapagliflozin (DAPA), for 4 weeks (raises blood ketones). Cardiac function was assessed by echocardiography. Cardiac energy metabolism was measured in isolated working hearts perfused with 5 mM glucose, 0.8 mM palmitate, and either 0.2 mM or 0.6 mM β-hydroxybutyrate (βOHB). ResultsTAC hearts had significantly decreased %EF compared to sham hearts, with no effect of DAPA. Glucose oxidation was significantly decreased in TAC hearts compared to sham hearts and did not decrease further in TAC hearts treated with high βOHB or in TAC DAPA hearts, despite βOHB oxidation rates increasing in both TAC vehicle and TAC DAPA hearts at high βOHB concentrations. Rather, increasing βOHB supply to the heart selectively decreased fatty acid oxidation rates. DAPA significantly increased ATP production at both βOHB concentrations by increasing the contribution of glucose oxidation to ATP production. ConclusionTherefore, increasing ketone concentration increases energy supply and ATP production in HFrEF without further impairing glucose oxidation.

Targeting a cardiac abundant and fibroblasts-specific piRNA (CFRPi) to attenuate and reverse cardiac fibrosis in pressure-overloaded heart failure

Cardiac fibrosis under chronic pressure overload is an end-stage adverse remodeling of heart. However, current heart failure treatments barely focus on anti-fibrosis and the effects are limited. We aimed to seek for a cardiac abundant and cardiac fibrosis specific piRNA, exploring its underlying mechanism and therapeutic potential. Whole transcriptome sequencing and the following verification experiments identified a highly upregulated piRNA (piRNA-000691) in transverse aortic constriction (TAC) mice, TAC pig, and heart failure human samples, which was abundant in heart and specifically expressed in cardiac fibroblasts. CFRPi was gradually increased along with the progression of heart failure, which was illustrated to promote cardiac fibrosis by gain- and loss-of-function experiments in vitro and in vivo. Knockdown of CFRPi in mice alleviated cardiac fibrosis, reversed decline of systolic and diastolic functions from TAC 6 weeks to 8 weeks. Mechanistically, CFRPi inhibited APLN, a protective peptide that increased in early response and became exhausted at late stage. Knockdown of APLN in vitro notably aggravated cardiac fibroblasts activation and proliferation. In vitro and in vivo evidence both indicated Pi3k-AKT-mTOR as the downstream effector pathway of CFRPi-APLN interaction. Collectively, we here identified CFPPi as a heart abundant and cardiac fibrosis specific piRNA. Targeting CFRPi resulted in a sustainable increase of APLN and showed promising therapeutical prospect to alleviate fibrosis, rescue late-stage cardiac dysfunction, and prevent heart failure.

CTRP3 alleviates mitochondrial dysfunction and oxidative stress injury in pathological cardiac hypertrophy by activating UPRmt via the SIRT1/ATF5 axis

Pathological cardiac hypertrophy is an independent risk factor for heart failure. Disruption of mitochondrial protein homeostasis plays a key role in pathological cardiac hypertrophy; however, the mechanism of maintaining mitochondrial homeostasis in pathological cardiac hypertrophy remains unclear. In this study, we investigated the regulatory mechanisms of mitochondrial protein homeostasis in pathological cardiac hypertrophy. Wildtype (WT) mice, knockout mice, and mice transfected with lentivirus overexpressing mouse C1q-tumor necrosis factor-related protein-3 (CTRP3) underwent transverse aortic constriction or sham surgery. After 4 weeks, cardiac function, mitochondrial function, and oxidative stress injury were examined. For mechanistic studies, neonatal rat cardiomyocytes were treated with small interfering RNA or overexpression plasmids for the relevant genes. CTRP3 overexpression attenuated transverse aortic constriction (TAC) induced pathological cardiac hypertrophy, mitochondrial dysfunction, and oxidative stress injury compared to that in WT mice. TAC or Ang II resulted in compensatory activation of UPRmt, but this was not sufficient to counteract pathologic cardiac hypertrophy. CTRP3 overexpression further induced activation of UPRmt during pathologic cardiac hypertrophy and thereby alleviated pathologic cardiac hypertrophy, whereas CTRP3 knockout or knockdown inhibited UPRmt. ATF5 was a key regulatory molecule of UPRmt, as ATF5 knockout prevented the cardioprotective effect of CTRP3 in TAC mice. In vitro, SIRT1 was identified as a possible downstream CTRP3 effector molecule, and SIRT1 knockout blocked the cardioprotective effects of CTRP3. Our results also suggest that ATF5 may be regulated by SIRT1. Our study demonstrates that CTRP3 activates UPRmt via the SIRT1/ATF5 axis under pathological myocardial hypertrophy, thus attenuating mitochondrial dysfunction and oxidative stress injury.

α-Ketoglutarate improves cardiac insufficiency through NAD+-SIRT1 signaling-mediated mitophagy and ferroptosis in pressure overload-induced mice

BackgroundIn heart failure (HF), mitochondrial dysfunction and metabolic remodeling lead to a reduction in energy productivity and aggravate cardiomyocyte injury. Supplementation with α-ketoglutarate (AKG) alleviated myocardial hypertrophy and fibrosis in mice with HF and improved cardiac insufficiency. However, the myocardial protective mechanism of AKG remains unclear. We verified the hypothesis that AKG improves mitochondrial function by upregulating NAD+ levels and activating silent information regulator 2 homolog 1 (SIRT1) in cardiomyocytes.MethodsIn vivo, 2% AKG was added to the drinking water of mice undergoing transverse aortic constriction (TAC) surgery. Echocardiography and biopsy were performed to evaluate cardiac function and pathological changes. Myocardial metabolomics was analyzed by liquid chromatography‒mass spectrometry (LC‒MS/MS) at 8 weeks after surgery. In vitro, the expression of SIRT1 or PINK1 proteins was inhibited by selective inhibitors and siRNA in cardiomyocytes stimulated with angiotensin II (AngII) and AKG. NAD+ levels were detected using an NAD test kit. Mitophagy and ferroptosis levels were evaluated by Western blotting, qPCR, JC-1 staining and lipid peroxidation analysis.ResultsAKG supplementation after TAC surgery could alleviate myocardial hypertrophy and fibrosis and improve cardiac function in mice. Metabolites of the malate-aspartate shuttle (MAS) were increased, but the TCA cycle and fatty acid metabolism pathway could be inhibited in the myocardium of TAC mice after AKG supplementation. Decreased NAD+ levels and SIRT1 protein expression were observed in heart of mice and AngII-treated cardiomyocytes. After AKG treatment, these changes were reversed, and increased mitophagy, inhibited ferroptosis, and alleviated damage in cardiomyocytes were observed. When the expression of SIRT1 was inhibited by a selective inhibitor and siRNA, the protective effect of AKG was suppressed.ConclusionSupplementation with AKG can improve myocardial hypertrophy, fibrosis and chronic cardiac insufficiency caused by pressure overload. By increasing the level of NAD+, the SIRT-PINK1 and SIRT1-GPX4 signaling pathways are activated to promote mitophagy and inhibit ferroptosis in cardiomyocytes, which ultimately alleviates cardiomyocyte damage.

Sacubitril/valsartan reduces proteasome activation and cardiomyocyte area in an experimental mouse model of hypertrophy

Sacubitril/valsartan (Sac/Val) belongs to the group of angiotensin receptor–neprilysin inhibitors and has been used for the treatment of heart failure (HF) for several years. The mechanisms that mediate the beneficial effects of Sac/Val are not yet fully understood. In this study we investigated whether Sac/Val influences the two proteolytic systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP), in a mouse model of pressure overload induced by transverse aortic constriction (TAC) and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) treated with endothelin-1 (ET1) serving as a human cellular model of hypertrophy. TAC mice showed a continuous decline in cardiac function starting from day 14 after surgery. Administration of Sac/Val for 6 weeks counteracted the deterioration of cardiac function and attenuated hypertrophy and fibrosis in TAC mice. The expression of ALP key markers did not differ between the groups. Proteasome activity was higher in TAC mice and normalized by Sac/Val. In hiPSC-CMs, all treatments (Sac, Val or Sac/Val) normalized mean cell area. However, Sac alone or in combination with Val, but not Val alone prevented ET1-induced hypertrophic gene program and proteomic changes. In conclusion, Sac/Val normalized proteasome activity, improved cardiac function and reduced fibrosis and hypertrophy in TAC mice. Molecular analysis in hiPSC-CMs suggests that a major part of the beneficial effects of Sac/Val is derived from the Sac action rather than from Val.

STING activation in cardiomyocytes drives hypertrophy-associated heart failure via NF-κB-mediated inflammatory response

Accumulating evidence highlights the key importance of innate immunity in heart hypertrophy and failure. Though stimulator of interferon genes (STING) is an integral innate immunity regulator, whether cardiomyocyte-derived STING driving cardiac hypertrophy and failure has rarely been explored, nor has its underlying mechanism been clarified. Herein, we addressed these two questions through several mouse experiments. Our results revealed that cardiac tissues from patients exhibiting cardiac hypertrophy markedly increased STING expression. Myocardial tissues of mice challenged with angiotensin II (Ang II) or transverse aortic constriction (TAC) also showed that STING was consistently upregulated and activated. Activation of STING by cGAMP or DMXAA resulted in cardiomyocyte hypertrophy in vitro, which was abolished by STING knockout. Furthermore, deleting or pharmacologically inhibiting STING attenuated cardiac hypertrophy and dysfunction in TAC or Ang II-treated mice. In contrast, mice with cardiomyocyte-specific STING activation developed cardiac hypertrophy and failure. Mechanistically, NF-κB signaling but not TBK1 or autophagy formation was implicated in STING -induced cardiac hypertrophy and failure. Collectively, we identified that STING-NF-κB axis mediated inflammatory response to drive cardiac hypertrophy-associated heart failure, highlighting its promise as a potential therapeutic target in clinical practice.

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation “two-pore channel” (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Betaine therapy of cardiac hypertrophy through inhibition of Hydroxy-carboxylic Acid Receptor 1 signaling

Abstract Background Hydroxy-carboxylic Acid Receptor 1 (HCAR1), formerly known as G protein-coupled Receptor 81 (GPR81), is a cell membrane receptor for lactate, and essential for activation of a variety of intracellular signaling pathways. We found a correlation between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy. However, the regulatory mechanism remains unclear. Purpose Our purpose is to investigate the regulatory mechanism between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy. Methods Neonatal rat cardiomyocytes (NRCMs) were used for preparing cardiac hypertrophy model in vitro by phenylephrine (PE) stimulation, and C57 BL/6 mice were used for preparing pressure overloaded-induced cardiac hypertrophy model in vivo by TAC (transverse aortic constriction) surgery. Adenovirus containing shRNA-HCAR1 (ADV-shHCAR1) were constructed to knockdown HCAR1 in NRCMs. Binding interaction between HCAR1 and Betaine was predicted by AUTODOCK 4 and further validated with RNA-seq analysis. Results Lactate concentration and HCAR1 expression were both increased in the NRCMs upon PE treatment. Activation of HCAR1 by its agonist 3-Chloro-5-hydroxybenzoic acid (3Cl-HBA) specially induced hypertrophy in NRCMs. Furthermore, the hypertrophy phenotypes in NRCMs showed alleviation by transfection of ADV-shHCAR1. An AI-based molecule-docking screening analysis identified Betaine as a potential molecule to bind HCAR1. Treatment with Betaine significantly attenuated PE- or 3Cl-HBA- induced hypertrophy in NRCMs in vitro. In addition, Betaine administration of TAC mice (i.g. 50 mg/kg/day) for 4 weeks in vivo led to increase of ejection fraction (EF) and decreases of left ventricular (LV) posterior wall thickness, heart weight/tibia length ((HW/TL), cross-sectional area and cardiac fibrosis area as well. RNA-seq analysis and functional assays demonstrated that Betaine could bind to HCAR1, activated Hippo signaling pathway, and thereby blocked the HCAR1-mediated hypertrophy in cardiomyocytes. Conclusions In summary, the current study not only identified HCAR1 as a therapeutic target of cardiac hypertrophy, but also demonstrated Betaine as a therapeutic candidate molecule holding potential to benefit cardiac hypertrophy patients.

Abstract 14400: Tipifarnib Mediated Reduction of Circulating Exosomes Inhibit Pressure Overload-Induced Cardiac Remodeling and Hypertrophy

Circulating extracellular vesicles, including exosomes, and their content have been implicated in cardiac tissue remodeling during cardiovascular disease progression. However, their role in cardiac hypertrophy and heart failure is very limited. Therefore, we investigated the role of circulating exosomes in cardiac dysfunction and remodeling in a transverse aortic constriction (TAC) model of heart failure using a recently identified exosome biogenesis inhibitor, Tipifarnib. Ten-week-old C57BL6J male mice were randomized into three groups i.e., Sham, TAC and Tipifarnib treated (10 mg/kg) TAC (TAC-Tip). The untreated TAC mice showed a significant increase in circulating exosomes along with gradual development of cardiac hypertrophy accompanied with reduced cardiac functions and increased fibrosis. On the contrary, TAC-Tip mice showed a significant reduction in circulating exosomes and remarkably improved cardiac left ventricular functions, reduced cardiac hypertrophy, fibrosis, and systemic inflammation. Strikingly, when primary mouse cardiac fibroblast cells were treated with exosomes derived from serum of TAC mice, the fibrosis related genes expression was significantly amplified compared to those treated with exosomes from TAC-Tip mice serum. Further, systemic injections of TAC-serum exosomes in normal, uninjured mice for 8 weeks, reduced cardiac function and increased cardiomyocyte hypertrophy. Small RNA sequencing analysis of serum exosomes from TAC and TAC-Tip mice displayed an extensively differential exosomal miRNAs profile. To evaluate the crucial role of miRNA in the circulating exosomes, we selected miR 331-5p based on the most differentially expressed miR and validated its expression in heart tissue and serum exosomes of Sham, TAC and TAC-Tip mice. Functional studies showed miR 331-5p inhibited HOXC8, a critical regulator of fibrosis. Overall, our studies reveal a mechanism by which treatment of Tipifarnib in TAC mice, alters not just the exosome numbers but also circulating exosomes content including miR 331-5p that directly inhibits HOXC8 and reduces fibrosis. Thus, Tipifarnib mediated inhibition of circulating exosomes may serve as a viable strategy to prevent progressive cardiac remodeling to heart failure.

Abstract 16785: Hypertension Induces Metabolite Content Alterations in Hypoperfused Mouse Brain Cortex

Introduction and aims: Hypertension is one of the main risk factors for dementias. To understand the link between vasculature and neuronal dysfunction in hypertension-induced cognitive impairment we leverage MRI, a translational imaging tool that let us concomitantly analyze cerebral structure, function and metabolism. Methods: Transverse aortic constriction (TAC) was performed in C57Bl/6J mice to induce cerebral hypertension. The brain was characterized by structural MRI, cerebral blood flow (CBF) was analyzed by perfusion MRI and cortical cerebral metabolism was characterized by point-resolved spectroscopy performed on a small-animal dedicated 7T MRI. Finally, we characterized the microvascular structure in regions impacted by hypertension by immunofluorescence. Results: TAC mice shown hemodynamic alterations in cortical CBF compared to sham mice (A). Macroscopic volumetric analyses of the cortex shown no significant alterations, however hypoperfused cortical regions shown local cortical thickening, suggesting a local swelling due to inflammatory processes (B). The histological microvascular analysis evidenced capillary Blood-Brain Barrier loss of integrity (C). Finally, this cortical region shown alterations in glucose and glutamine metabolite concentration measured by MR spectroscopy (D). Conclusions: Leveraging MRI we have concomitantly characterized the cortical structure, function and metabolism in TAC-hypertensive mice. These alterations in cerebral blood flow and metabolism of glucose/glutamine are biomarkers of ongoing cerebral injury and are potential targets of further investigation to shed light on the interplay between vascular and neuronal injury in hypertension.

Abstract 15096: miR-132 Inhibition Shows Anti-Hypertrophic Effects in Preclinical Heart Failure Models and in Exploratory Pharmacodynamic Parameters of a First-in-Human Phase 1b Clinical Trial

Introduction: Cardiac expression levels of miR-132 are elevated in patients with heart failure (HF) and have been associated and provoke pathological cardiac hypertrophy. To inhibit these cardiac remodeling processes and restore normal cardiac function, we developed the synthetic antisense oligonucleotide inhibitor CDR132L targeting miR-132. Anti-hypertrophic effects of miR-132 inhibition have been shown previously in cell culture models and a mouse model of pressure overload-induced HF. Methods and Results: MiR-132 inhibition with CDR132L was now tested in a mouse model of pressure overload induced HF induced by transverse aortic constriction (TAC) and in three different porcine HF models to further confirm the translatability of therapeutic effects. The first pig model was an early post-myocardial infarction (MI). Pigs were treated with CDR132L by intravenous (IV) or intracoronary (IC) infusion on day 3 and day 28 and analyzed on day 56 post-MI. As a second model, chronic post-MI HF pigs received monthly IV treatments over 3 or 5 months starting from month 1 post-MI. Third, percutaneous aortic constriction was induced in pigs by reduction stent implantation and CDR132L was administered IC on day 0 and day 28 followed by analysis on day 56. Hypertrophy was determined by cell size measurements in wheat germ agglutinin (WGA) stained cardiac sections and was significantly decreased between 15% and 29% in CDR132L-treated groups with sufficient dose levels compared to placebo-treatment independent from the large animal model and dosing regimen. Furthermore, TAC mice treated with CDR132L showed a significant decrease in left ventricular mass by 14 % compared to placebo. In line with these data, the cardiac remodeling marker lipocalin-2 (NGAL) was reduced in plasma of chronic HF patients assessed in a first-in-human phase 1b clinical to test safety, efficacy, and also exploratory pharmacodynamics of CDR132L (NCT04045405). Conclusions: Our results confirm beneficial effects of CDR132L treatment on pathological cardiac remodeling in HF.

Abstract 16405: Perm1 Protects the Heart From Pressure Overload-Induced Dysfunction by Promoting Oxidative Metabolism

Introduction: Defects in ATP production lead to cardiac dysfunction and heart failure (HF) while restoring reduced mitochondrial (Mito) function mitigates HF. Previously we showed that a novel Mito protein, Perm1 (PGC-1 and ERR-induced regulator, muscle 1), promotes Mito biogenesis in cardiomyocytes (CM). Here we studied the role of myocardial Perm1 in vivo . Hypothesis: Perm1 protects the heart from pressure overload-induced dysfunction. Methods: First, we generated cardiac-specific Perm1 transgenic mice (Perm1cTG). Next, we induced pressure overload by transverse aortic constriction (TAC) or performed Sham operations on 10-week-old control (CTL) and Perm1cTG male mice. Heart function, histology, and biochemical analyses were performed eight weeks postoperatively. To understand the mechanism of Perm1, the heart protein samples were subjected to quantitative mass spectrometry (MS) analysis, and we also identified Perm1 binding proteins in the heart by MS. Finally, mouse neonatal CM were infected with adenoviruses expressing shMic60 (knock down) or shControl in combination with Perm1 or LacZ, followed by oxygen consumption rate (OCR) analysis. Results: Sham-operated Perm1cTG and CTL mice showed no differences in function and morphology. Following TAC, CTL developed severely reduced heart function, while Perm1cTG had only minimal change. Perm1cTG TAC mice also showed reduced cardiac hypertrophy, Anf and Bnp mRNA expression, and fibrosis, compared to CTL TAC, showing Perm1 protects the heart from hemodynamic stress. Perm1cTG TAC hearts showed significantly increased OCR compared to CTL TAC, suggesting Perm1 preserves TAC-induced CM Mito dysfunction. MS analysis identified MICOS (mitochondrial contact site and cristae organizing system) and oxidative phosphorylation proteins were upregulated in Perm1cTG TAC hearts compared to CTL TAC. Moreover, we found that Perm1 binds to Mic60, which is a core component of the MICOS. Finally, Mic60 was required for Perm1-induced maximal oxidative capacity, suggesting Perm1 acts through Mic60. Conclusion: Perm1 protects the heart against pressure overload by restoring oxidative metabolism. Future studies will reveal the role of Perm1/Mic60 in the heart. This work is supported by NIH R01HL151239.

Circular RNA circSMAD4 regulates cardiac fibrosis by targeting miR-671-5p and FGFR2 in cardiac fibroblasts

Heart failure is a leading cause of death and is often accompanied by activation of quiescent cardiac myofibroblasts, which results in cardiac fibrosis. In this study, we aimed to identify novel circular RNAs that regulate cardiac fibrosis. We applied transverse aortic constriction (TAC) for 1, 4, and 8weeks in mice. RNA sequencing datasets were obtained from cardiac fibroblasts isolated by use of a Langendorff apparatus and then further processed by use of selection criteria such as differential expression and conservation in species. CircSMAD4 was upregulated by TAC in mice or by transforming growth factor (TGF)-β1 in primarily cultured human cardiac fibroblasts. Delivery of si-circSMAD4 attenuated myofibroblast activation and cardiac fibrosis in mice treated with isoproterenol (ISP). si-circSmad4 significantly reduced cardiac fibrosis and remodeling at 8weeks. Mechanistically, circSMAD4 acted as a sponge against the microRNA miR-671-5p in a sequence-specific manner. miR-671-5p was downregulated during myofibroblast activation and its mimic form attenuated cardiac fibrosis. miR-671-5p mimic destabilized fibroblast growth factor receptor 2 (FGFR2) mRNA in a sequence-specific manner and interfered with the fibrotic action of FGFR2. The circSMAD4-miR-671-5p-FGFR2 pathway is involved in the differentiation of cardiac myofibroblasts and thereby the development of cardiac fibrosis.

Exploring key genes associated with neutrophil function and neutrophil extracellular traps in heart failure: a comprehensive analysis of single-cell and bulk sequencing data

Background: Heart failure (HF) is a complex and heterogeneous manifestation of multiple cardiovascular diseases that usually occurs in the advanced stages of disease progression. The role of neutrophil extracellular traps (NETs) in the pathogenesis of HF remains to be explored.Methods: Bioinformatics analysis was employed to investigate general and single-cell transcriptome sequencing data downloaded from the GEO datasets. Differentially expressed genes (DEGs) associated with NETs in HF patients and healthy controls were identified using transcriptome sequencing datasets and were subsequently subjected to functional enrichment analysis. To identify potential diagnostic biomarkers, the random forest algorithm (RF) and the least absolute shrinkage and selection operator (LASSO) were applied, followed by the construction of receiver operating characteristic (ROC) curves to assess accuracy. Additionally, single-cell transcriptome sequencing data analysis identified key immune cell subpopulations in TAC (transverse aortic constriction) mice potentially involved in NETs regulation. Cell-cell communication analysis and trajectory analysis was then performed on these key cell subpopulations.Results: We identified thirteen differentially expressed genes (DEGs) associated with NET through differential analysis of transcriptome sequencing data from HF (heart failure) samples. Utilizing the Random Forest and Lasso algorithms, along with experimental validation, we successfully pinpointed four diagnostic markers (CXCR2, FCGR3B, VNN3, and FPR2) capable of predicting HF risk. Furthermore, our analysis of intercellular communication, leveraging single-cell sequencing data, highlighted macrophages and T cells as the immune cell subpopulations with the closest interactions with neutrophils. Pseudo-trajectory analysis sheds light on the differentiation states of distinct neutrophil subpopulations.Conclusion: In this study, we conducted an in-depth investigation into the functions of neutrophil subpopulations that infiltrate cardiac tissue in TAC mice. Additionally, we identified four biomarkers (CXCR2, FCGR3B, VNN3, and FPR2) associated with NETs in HF. Our findings enhance the understanding of immunology in HF.

Stachydrine Hydrochloride Regulates the NOX2-ROS-Signaling Axis in Pressure-Overload-Induced Heart Failure.

Our previous studies revealed the protection of stachydrine hydrochloride (STA) against cardiopathological remodeling. One of the underlying mechanisms involves the calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII). However, the way STA influences CaMKII needs to be further investigated. The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-coupled reactive oxygen species (ROS) overproduction putatively induces the oxidative activation of CaMKII, resulting in the occurrence of pathological cardiac remodeling and dysfunction in experimental models of mice. Thus, in this study, we assessed the role of the NOX2-ROS signal axis in STA cardioprotection. The transverse aortic constriction (TAC)-induced heart failure model of mice, the phenylephrine-induced hypertrophic model of neonatal rat primary cardiomyocytes, and the H2O2-induced oxidative stress models of adult mouse primary cardiomyocytes and H9c2 cells were employed. The echocardiography and histological staining were applied to assess the cardiac effect of STA (6 mg/kg/d or 12 mg/kg/d), which was given by gavage. NOX2, ROS, and excitation-contraction (EC) coupling were detected by Western blotting, immunofluorescence, and calcium transient-contraction synchronous recordings. ROS and ROS-dependent cardiac fibrosis were alleviated in STA-treated TAC mice, demonstrating improved left ventricular ejection fraction and hypertrophy. In the heart failure model of mice and the hypertrophic model of cardiomyocytes, STA depressed NOX2 protein expression and activation, as shown by inhibited translocation of its phosphorylation, p67phox and p47phox, from the cytoplasm to the cell membrane. Furthermore, in cardiomyocytes under oxidative stress, STA suppressed NOX2-related cytosolic Ca2+ overload, enhanced cell contractility, and decreased Ca2+-dependent regulatory protein expression, including CaMKⅡ and Ryanodine receptor calcium release channels. Cardioprotection of STA against pressure overload-induced pathological cardiac remodeling correlates with the NOX2-coupled ROS signaling cascade.

Alamandine attenuates oxidative stress in the right carotid following transverse aortic constriction in mice

ObjectivePressure overload can result in significant changes to the structure of blood vessels, a process known as vascular remodeling. High levels of tension can cause vascular inflammation, fibrosis, and structural alterations to the vascular wall. Prior research from our team has demonstrated that the oral administration of alamandine can promote vasculoprotective effects in mice aorta that have undergone transverse aortic constriction (TAC). Furthermore, changes in local hemodynamics can affect the right and left carotid arteries differently after TAC. Thus, in this study, we aimed to assess the effects of alamandine treatment on right carotid remodeling and the expression of oxidative stress-related substances induced by TAC. Methods and resultsMale C57BL/6 mice were categorized into three groups: Sham, TAC, and TAC treated with alamandine (TAC+ALA). Alamandine treatment was administered orally by gavage (30 µg/kg/day), starting three days before the surgery, and continuing for a period of fourteen days. Morphometric analysis of hematoxylin and eosin-stained sections revealed that TAC induced hypertrophic and positive remodeling in the right carotid artery. Picrosirius Red staining also demonstrated an increase in total collagen deposition in the right carotid artery due to TAC-induced vascular changes. Alamandine treatment effectively prevented the increase in reactive oxygen species production and depletion of nitric oxide levels, which were induced by TAC. Finally, alamandine treatment was also shown to prevent the increased expression of nuclear factor erythroid 2-related factor 2 and 3-nitrotyrosine that were induced by TAC. ConclusionOur results suggest that alamandine can effectively attenuate pathophysiological stress in the right carotid artery of animals subjected to TAC.

HDAC5 inhibition attenuates ventricular remodeling and cardiac dysfunction

BackgroundThis study aimed to investigate the role of histone deacetylase 5 (HDAC5) in ventricular remodeling and explore the therapeutic potential of the HDAC5 inhibitor LMK235.MethodsA transverse aortic constriction (TAC) mouse model and angiotensin II (Ang II)-treated H9C2 cells were used to evaluate the effects of HDAC5 inhibition with LMK235 on ventricular remodeling and cardiac dysfunction. Additionally, the involvement of the extracellular signal-regulated kinase (ERK)/early growth response protein 1 (EGR1) signaling pathway in regulating myocyte enhancer factor 2 A (MEF2A) expression was assessed.ResultsHDAC5 was upregulated in TAC mice and Ang II-treated H9C2 cells, suggesting its involvement in ventricular remodeling and cardiac dysfunction. LMK235 treatment significantly improved cardiac function in TAC mice and attenuated TAC-induced ventricular remodeling and Ang II-induced H9C2 cell hypertrophy. Mechanically, HDAC5 inhibition activated the ERK/EGR1 signaling pathway.ConclusionsOur findings demonstrate that HDAC5 may suppress the activation of ERK/EGR1 signaling to regulate MEF2A expression and therefore participate in cardiac pathophysiology.