Circulation Research

The adult mammalian heart has a limited ability to regenerate lost myocardium following myocardial infarction (MI), largely due to the poor proliferative capacity of cardiomyocytes. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a known regulator of cell quiescence, though the mechanisms underlying its function remain unclear. Previous studies have shown that pharmacological inhibition of DYRK1A using harmine induces cardiomyocyte cell cycle re-entry after ischemia/reperfusion (I/R) MI. Here, we developed a computational network model of DYRK1A-mediated regulation of the cell cycle, which predicts how DYRK1A inhibition promotes cardiomyocyte re-entry. To validate these predictions, we tested selective DYRK1A inhibitors and observed robust induction of cell cycle activity in neonatal rat cardiomyocytes (NRCMs). Integrating our network model with bulk RNA-sequencing data from DYRK1A inhibitor-treated NRCMs, we identified E2F1 as a key transcriptional driver of cell cycle gene expression. Finally, we demonstrate that both pharmacological and post-developmental inhibition of DYRK1A enhances heart function and increases cardiomyocyte cycling following I/R MI. Our findings suggest that functional recovery induced by small molecule inhibitor of DYRK1A is mediated by the induction of cycling cardiomyocytes. One Sentence SummaryInhibition of DYRK1A through LCTB-92 induces cardiomyocyte cycling and improved heart function in a mouse model of ischemic/reperfusion injury.

We address a paradox, that AMPK may facilitate hypoxic pulmonary vasoconstriction and its deficiency precipitate pulmonary hypertension. Here we show that AMPK-1/2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the newborn. Accordingly, dual AMPK-1/2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularization and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no edema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes are markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation is also evident. Importantly, there is no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction is attenuated by AMPK-1 or AMPK-2 deletion without triggering pulmonary hypertension.

ObjectiveTo investigate the effects of carnosine on heart failure and to examine whether this is associated with reduced immunogenicity of oxidatively-generated aldehyde modified proteins. BackgroundHeart failure is associated with the accumulation of lipid derived aldehydes that form immunogenic protein adducts. However, the pathological impact of these aldehydes and aldehyde-modified proteins in heart failure has not been assessed. Histidyl dipeptides, such as carnosine found in the heart, bind to aldehydes, and their protein adducts. However, the effects of carnosine on heart failure or the antigenicity of aldehyde modified proteins have not been studied. MethodsMale, wild type C57BL/6J mice were subjected to either sham or transverse aortic constriction (TAC) surgery. To increase carnosine levels, they were placed on drinking water with or without {beta}-alanine prior to surgery, and for the remainder of the study. Cardiac function was evaluated by echocardiography, and the levels of histidyl dipeptides, immune cell populations, and CD4+ T cell activation were assessed via LC-MS/MS and flow cytometry, respectively. ResultsMyocardial levels of histidyl dipeptides decreased at both 3- and 8-weeks post-TAC. Supplementation with {beta}-alanine increased myocardial histidyl dipeptide levels, attenuated adverse cardiac remodeling, and reduced aldehyde stress. Carnosine formed covalent bond with protein-bound aldehydes in the failing heart, reducing their antigenic potential and decreasing activation of dendritic cells and CD4+ T cells in vitro. {beta}-alanine supplementation decreased the population of CD11b+CD64-Ly6G+ neutrophils and CD4+ CD44+ effector T cells in the failing heart. ConclusionsIncreasing myocardial carnosine levels reduces aldehyde stress, dampens maladaptive immune responses, and preserves cardiac function during heart failure. HIGHLIGHTSO_LILevels of endogenous dipeptide carnosine are depleted in failing hearts, while supplementation of the carnosine precurson {beta}-alanine increases myocardial carnosine and preserves cardiac function during heart failure. C_LIO_LIHeart failure is associated with increased activation and infiltration of CD4+ T cells and generation of aldehyde modified protein adducts in failing hearts. C_LIO_LIThe free aldehyde moiety of aldehyde modified protein adducts activates CD4+ T cells through dendritic cell presentation and capping this moiety with carnosine diminishes their antigencity. C_LIO_LIIncreasing myocardial carnosine levels diminishes aldehyde stress and activation of CD4+ T cells during heart failure. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/671840v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@28fc7corg.highwire.dtl.DTLVardef@d851ccorg.highwire.dtl.DTLVardef@1e24a5dorg.highwire.dtl.DTLVardef@18023b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome with incompletely understood molecular mechanisms. Histone serotonylation is a recently identified epigenetic modification in which serotonin is covalently conjugated to glutamine 5 of histone H3 in H3K4me3-marked nucleosomes. Here, we investigated the role of transglutaminase 2 (TGM2)-mediated histone serotonylation in HFpEF. In a mouse model of HFpEF induced by salty drinking water, unilateral nephrectomy and aldosterone infusion (SAUNA), cardiac H3K4me3Q5ser and nuclear TGM2 levels were increased. Cardiomyocyte-specific TGM2-deficient mice developed aggravated HFpEF phenotypes after SAUNA exposure, including worsened diastolic dysfunction, reduced exercise capacity, pulmonary congestion and delayed cardiomyocyte relaxation. CUT&RUN sequencing identified H3K4me3Q5ser-enriched regions predominantly around transcription start sites after SAUNA exposure, with notable enrichment at genes associated with G2/M checkpoint-related stress-response signaling. RNA sequencing further showed that activation of this pathway was impaired in SAUNA-exposed TGM2-deficient hearts. In cardiac myocytes, calcium-binding sites and nuclear localization of TGM2 support checkpoint-related stress-response gene activation in cardiac myocytes. Pharmacological WEE1 inhibition, which activates downstream CDK1-associated checkpoint signaling, partially rescued the aggravated HFpEF phenotype in TGM2-deficient mice. Finally, in patients with HFpEF, lower circulating serotonin levels were associated with adverse cardiac outcomes, and cardiomyocyte H3K4me3Q5ser levels correlated with serum serotonin concentrations. These findings suggest that cardiomyocyte TGM2-mediated histone serotonylation represents a stress-adaptive, cardioprotective epigenetic mechanism in HFpEF.

Cardiovascular disease remains the leading cause of death globally, underscoring the need for physiologically relevant models to investigate mechanisms of heart failure and arrhythmia. Chronic activation of hypoxic signaling pathways, particularly via the hypoxia-inducible factor (HIF) axis, is a key contributor to cardiac remodeling under stress. A major regulator of HIF signaling is the von Hippel-Lindau tumor suppressor (VHL) which, under normoxic conditions, targets HIF for proteasomal degradation. Loss of VHL results in HIF accumulation and persistent hypoxic signaling, but constitutive cardiomyocyte-specific Vhl knockout models are confounded by developmental effects and early mortality. Here, we develop and characterize an inducible, cardiomyocyte-specific Vhl knockout mouse model as a non-invasive and temporally controlled system to study chronic hypoxic stress and its contribution to cardiac remodeling and disease. VhlLoxP/LoxP;MHC-MerCreMer+/- mice were administered tamoxifen to induce Vhl deletion in adult cardiomyocytes. Within 5-7 days post-induction, mice displayed reduced ejection fraction, increased cardiac diameter, and elevated expression of cardiac stress markers. Transcriptomic and protein analyses revealed downregulation of key genes involved in cardiac structure and electrophysiology, including Gja1 (Cx43), Cdh2 (N-cadherin), Cacna1c (CaV1.2), and Kcnq1. Importantly, these changes preceded overt cardiac remodeling, as confirmed in an abbreviated tamoxifen protocol. This inducible Vhl knockout model recapitulates hallmark features of dilated cardiomyopathy and highlights a subset of cardiac structural and ion channel genes as sensitive early responders to chronic hypoxic stress. This platform enables mechanistic dissection of disease onset and progression in ischemic heart disease and serves as a well-controlled and reproducible model for evaluating novel therapeutic strategies.

Symptomatic heart failure (sHF) with cardiac dysfunction, edema, and mortality are driven by overactivation of the renin-angiotensin-aldosterone system (RAAS). Renin is widely recognized as a key initiator of RAAS function, yet the mechanisms that activate renin remain a mystery. We discovered that activated coagulation factor XII generates active renin in the circulation and is directly linked to pathological activation of the systemic RAAS, development of sHF, and increased mortality. These findings suggest a new paradigm for therapeutically modulating the RAAS in sHF and other pathological conditions.

BackgroundWhile cardiomyocytes undergo terminal differentiation postnatally and rarely re-enter the cell cycle, the endogenous mechanisms that propagate differentiation and prevent de-differentiation remain unclear. The monomeric heme protein myoglobin, which stores oxygen and regulates reactive oxygen/nitrogen species balance in the heart, increases in expression by over 50% during cardiomyocyte differentiation. Though myoglobin deletion without significant compensation is embryonic lethal in mice, a role for the protein in regulating cardiomyocyte differentiation has not been tested. We hypothesized that myoglobin expression is required for cardiomyocyte differentiation and the loss of myoglobin enables de-differentiation. MethodsMyoglobin was genetically silenced in HL-1, H9C2 cells, and neonatal rat ventricular cardiomyocytes (NRVM) to examine myoglobin-dependent effects on differentiation, proliferation, and Hippo pathway signaling. A zebrafish model of Mb depletion was made using CRISPR-Cas9 to test the effect of myoglobin depletion on cardiac regeneration after apical resection injury in vivo. ResultsMyoglobin deletion in cultured cell lines and NRVM decreased the gene expression of cardiomyocyte differentiation markers (troponin, myosin light chain, and myosin heavy chain), upregulated markers of dedifferentiation (runx1 and dab2) and stimulated cell proliferation. Mechanistically, we show that the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, which activates the enzyme to phosphorylate the downstream Yes-associated protein (YAP) transcription factor, which prevents its transcriptional activity. Thus, the loss of myoglobin results in the de-phosphorylation and nuclear translocation of YAP, which propagates proliferation and fetal gene expression. In vivo, myoglobin-deficient zebrafish hearts recapitulated the increase in YAP signaling and showed accelerated regeneration at 20 days post apical injury. ConclusionWe a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation, and a regulator of the Hippo pathway. These findings suggest myoglobin as a potential target for strategies to enhance cardiac development and improve cardiac repair and regeneration.

RationaleThe most significantly associated atrial fibrillation (AF) risk loci in humans map to a noncoding gene desert upstream of the evolutionarily conserved left-right (LR) transcription factor Pitx2, a master regulator of LR asymmetric organ development. Pitx2 dosage is fundamentally linked to the development of sinus node dysfunction (SND) and AF, the most common cardiac arrhythmia affecting adults, but the mechanistic basis for this remains obscure. We identified a conserved long noncoding RNA (lncRNA), Playrr, which is exclusively transcribed on the embryos right side, opposite to Pitx2 on the left, that participates in mutually antagonistic transcriptional regulation with Pitx2. ObjectiveThe objective of this study was to investigate a role of Playrr in regulating Pitx2 transcription and protecting against the development of cardiac rhythm disturbances. Methods and ResultsPlayrr expression in the developing heart was analyzed with RNA in situ hybridization. Playrr was expressed asymmetrically (on the right) to Pitx2 (on the left) in developing mouse embryos, including in mouse embryonic sinoatrial node cells. We utilized CRISPR/Cas9 genome editing in mice to target Playrr, generating mice lacking Playrr RNA transcript (PlayrrEx1sj allele). Using qRT-PCR we detected upregulation of the cardiac isoform, Pitx2c, during visceral organ morphogenesis in PlayrrEx1sj mutant embryos. Surface ECG (AliveCor(R)) and 24-hour telemetry ECG detected bradycardia and irregular interbeat (R-R) intervals suggestive of SND in PlayrrEx1sj mutant adults. Programmed stimulation of PlayrrEx1sj mutant adults resulted in pacing-induced AF. Within the right atrium of PlayrrEx1sj mutant hearts, Massons trichrome stain revealed increased collagen deposition indicative of fibrosis, and immunofluorescence demonstrated mis-localization of Connexin 43 in atrial cardiomyocytes. These findings suggested an altered atrial substrate in PlayrrEx1sj adult mice. Finally, transcriptomic analysis by chromatin run-on and sequencing (ChRO-seq) in atria of PlayrrEx1sj mutant mice compared to wild type controls revealed differential expression of genes involved in cell-cell adhesion and motility, fibrosis, and dysregulation of the key cardiac genes Tbx5 and Hcn1. ConclusionsAdult mice lacking functional Playrr lncRNA transcript have baseline bradyarrhythmia and increased susceptibility to AF. These cardiac phenotypes are similar to those observed in Pitx2 heterozygous mice. Interactions between Pitx2 and Playrr may provide a genetic mechanism for modulating Pitx2 dosage and susceptibility to SND and AF.

BackgroundDepending upon the type of pathological stress, the heart undergoes concentric or eccentric remodeling. This structural change is associated with diastolic and/or systolic ventricular dysfunction reflecting differentially altered cardiomyocyte morphology, ultrastructure, metabolism, contractility, and survival, as well as interstitial myocardial fibrosis. Despite an association of both concentric and eccentric remodeling with heart failure and sudden death, the molecular mechanisms resulting in abnormal cardiac geometry remain poorly understood. A better understanding of the basic mechanisms conferring these contrasting forms of remodeling should inform novel approaches to preserve normal cardiac structure and function in cardiovascular disease. The protein phosphatase Cell Division Cycle 14A (CDC14A) and its substrate the lysine methyltransferase KMT5A are identified herein as key regulators of the balance between concentric and eccentric pathological cardiac remodeling. MethodsThe regulation of adult rat ventricular myocyte morphology by CDC14A and KMT5A was studied in vitro following gain and loss of function by expression of wild-type and mutant proteins and RNA interference (RNAi). Epigenomic regulation by KMT5A was studied by mapping histone 4 lysine 20 mono-methylation (H4K20me1) modified chromatin sites and correlating them with gene transcription. Regulation of pathological cardiac remodeling in vivo was demonstrated by CDC14A and KMT5A RNAi using adeno-associated virus (AAV) mediated cardiomyocyte-specific small hairpin RNA (shRNA) expression in mice. ResultsCDC14A inhibited the growth in width of cultured adult myocytes stimulated by -adrenergic receptor activation or by serum response factor. KMT5A was downregulated by CDC14A in cardiomyocytes and was required for myocyte growth in width. -adrenergic stimulation of KMT5A-dependent H4K20 mono-methylation across transcription units correlated with regulation of gene transcription. Accordingly, AAV-expressed KMT5A shRNA induced eccentric remodeling and cardiac dysfunction in wild-type mice. Conversely, expression of Cdc14A shRNA improved systolic function and cardiac structure and inhibited pathological gene expression in the Tpm1 E54K mouse with Dilated Cardiomyopathy. ConclusionsCDC14A-KMT5A-dependent epigenomic regulation of gene transcription constitutes a molecular switch that determines concentric versus eccentric cardiac remodeling. These findings identify CDC14A as a potential therapeutic target for the treatment of dilated cardiomyopathy and other forms of heart failure with reduced ejection fraction. Clinical PerspectiveO_ST_ABSWhat is newC_ST_ABSO_LIA function is identified for the first time for the protein phosphatase CDC14A in the heart, regulation of cardiomyocyte morphology and overall cardiac geometry in pathological cardiac remodeling. C_LIO_LIThe lysine methyltransferase KMT5A is shown to mediate the effects of CDC14A in the adult cardiomyocyte by regulating H4K20 mono-methylation, such that reduced KMT5A expression promotes a phenotype resembling Dilated Cardiomyopathy. C_LIO_LIH4K20me1 epigenomic modification is identified as a regulator of cardiac structure and function. C_LI Clinical implicationsO_LICDC14A loss of function experimentation in vivo, resulting in improved cardiac structure and function in a mouse model of Dilated Cardiomyopathy, suggests that CDC14A is a novel therapeutic target for heart failure with reduced ejection fraction. C_LI

BackgroundWildland firefighters experience repeated occupational exposure to wildfire smoke at high particulate matter (PM) concentrations, leading to elevated cardiovascular disease risk and hypertension prevalence. However, the pathophysiological processes linking cumulative smoke inhalation to vascular damage and blood pressure elevation remain poorly characterized. To evaluate these effects under controlled exposure conditions, we used a preclinical exposure model calibrated to match the cumulative PM burden deposited in wildland firefighter airways over 7-14 years of service. Male apolipoprotein E knockout (Apoe-/-) mice underwent whole-body inhalation of Douglas fir smoke or filtered air for 2 hours/day, 5 days/week, for 8 or 16 weeks at target PM concentrations of 40 mg/m3. ResultsProlonged smoke exposure induced sustained elevation of circulating tumor necrosis factor-alpha (TNF-), interleukin-1 beta (IL-1{beta}), and interleukin-6 (IL-6), coupled with diffused nuclear factor kappa B (NF-{kappa}B) activation throughout the aortic wall. Smoke inhalation disrupted endothelial adherens junctions, upregulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and promoted monocyte recruitment to aortic tissues, concurrent with enhanced monocyte chemoattractant protein-1 (MCP-1) expression. Oxidative stress was evidenced by increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 2 (NOX2) expression, elevated superoxide levels, and endothelial nitric oxide synthase (eNOS) uncoupling in the aorta, leading to lipid peroxidation and accompanied by intimal apoptosis. These inflammatory and oxidative perturbations occurred alongside a pro-fibrotic phenotypic shift characterized by transforming growth factor beta 1 (TGF-{beta}1) upregulation, myofibroblast differentiation, and progressive collagen accumulation in medial and adventitial compartments of the aortic wall. Functionally, smoke exposure progressively impaired aortic cyclic distensibility through combined wall thickening and circumferential tissue stiffening, while severely attenuating endothelium-dependent and nitric oxide (NO)-mediated vasodilation. These functional and structural shifts culminated in elevated systolic and diastolic blood pressures. While endothelial dysfunction reached maximal impairment by 8 weeks, aortic stiffening continued to worsen through 16 weeks of exposure, demonstrating differential temporal progression of vascular damage. ConclusionsThese findings demonstrate that occupationally relevant wildfire smoke exposure produces convergent inflammatory, oxidative, and profibrotic vascular remodeling with progressive loss of arterial compliance and impaired endothelium-dependent vasodilation, underscoring potential vascular targets for cardiovascular health surveillance and risk mitigation in wildland firefighters.

BackgroundMutation of the PRDM16 gene has been associated with human cardiomyopathy. The PRDM16 protein is a transcriptional regulator affecting cardiac development via Tbx5 and Hand1 regulating myocardial structure. Biallelic Prdm16 inactivation induces severe cardiac dysfunction with postnatal lethality and hypertrophy in mice. Early pathological events upon Prdm16 inactivation have not been explored. MethodsThis study performed in depth pathophysiological and molecular analysis of male and female Prdm16csp1/wt mice carrying systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes with transcriptomics, proteomics, and metabolomics. Kinetic modelling of the cardiac metabolism was undertaken in silico with CARDIOKIN. ResultsPrdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt hearts demonstrate moderate alterations of specific transcripts and protein levels with consistent upregulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre B-cell leukemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional upregulation was detected for Prdm16 itself probably by an autoregulatory mechanism. Prdm16csp1/wt cardiac tissue showed reduction of metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and tricarboxylic acid cycle. Global lipid metabolism was also affected with accumulation of triacylglycerides detected in male Prdm16csp1/wt hearts. In addition, Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. Metabolic modelling in silico suggested lowered fatty acid utilization in male and reduced glucose utilization in female Prdm16csp1/wt cardiac tissue. ConclusionsMonoallelic Prdm16 mutation restricts cardiac performance in Prdm16csp1/wt mice. Metabolic alterations precede transcriptional dysregulation in Prdm16csp1/wt cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype indicating a sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in PRDM16 associated cardiac pathology. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABS- Mutation of the PRDM16 gene has been associated with human cardiomyopathy. - Biallelic inactivation of Prdm16 in mice induces severe cardiac dysfunction with early postnatal lethality. - Prdm16 cooperates with transcription factors such as Tbx5 and Hand1 to activate transcriptional programs that define the development of the compacted myocardium. What New Information Does This Article Contribute?- Systemic, monoallelic inactivation in Prdm16csp1/wt mice induces cardiac dysfunction with normal survival. - Metabolic alterations are the leading pathophysiological consequences and induce cardiac hypoplasia. On the molecular level this is associated with upregulation of metabolic regulators Pyroxd2 and Pbxip1. - Metabolic response after Prdm16 inactivation occurs in a sex specific manner.

BACKGROUNDChanges in mitochondria have been implicated in atrial fibrillation (AF), but their manifestations and significance are poorly understood. Here, we studied changes in mitochondrial morphology and function during AF and assessed the effect of a mitochondrial-targeted intervention in a large animal model. METHODS AND RESULTSAtrial cardiomyocytes (ACMs) were isolated from dogs in electrically-driven AF for periods of 24 hours to 3 weeks and from humans with/without longstanding persistent AF. Mitochondrial Ca2+-concentration ([Ca2+]Mito), reactive oxygen species (mtROS) production, membrane potential ({Delta}{Psi}m), permeability transition-pore (mPTP) opening and flavin adenine dinucleotide (FAD) were measured via confocal microscopy; nicotine adenine dinucleotide (NADH) under ultraviolet light. mtROS-production increased within 24 hours and superoxide-dismutase type-2 was significantly reduced from 3-day AF. [Ca2+]Mito and mPTP-opening frequency/duration increased progressively during AF. Mitochondrial depolarization was detectable 24 hours after AF-onset. NADH increased by 15% at 24-hour AF, concomitant with increased pyruvate-dehydrogenase expression, then gradually decreased. Mitochondria enlarged and elongated at 24-hour and 3-day AF, followed by progressive fragmentation, rupture and shrinkage. Mitochondrial fusion protein-1 (MFN1) was reduced from 3-day to 3-week AF and phosphorylated dynamin-related protein-1 (p-DRP1ser-616) increased after 1 week of canine AF and in human AF. Addition of the mitochondrial antioxidant MitoTempo attenuated action-potential shortening and L-type Ca2+-current (ICaL)-downregulation in canine and human AF ACMs in vitro. Administration of the orally-active mitochondrial-targeted ubiquinone mitoquinone to dogs during 3-week AF prevented mitochondrial Ca2+-overload, mtROS-overproduction, structural damage and abnormalities in {Delta}{Psi}m and respiration. Functionally, mitoquinone reduced AF-induced Ca2+-current downregulation, action-potential abbreviation, contractile dysfunction and fibrosis, preventing AF-substrate development and AF-sustainability. CONCLUSIONSMitochondria show a series of changes during AF, with early hyperfunction and enhanced ROS-generation, followed by progressive damage and dysfunction. Mitochondrial-targeted therapy prevents mitochondrial dysfunction and attenuates adverse AF-related remodeling, positioning mitochondrial protection as a potential novel therapeutic target in AF.

BACKGROUNDCirculating levels of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates plasma cholesterol content by degrading LDL receptor, are correlated with the risk of acute myocardial infarction (AMI). Recent studies suggested that PCSK9 improves cardiac function beyond its effects on LDL cholesterol levels after cardiac ischemic injury, but its precise mechanism remains unclear. METHODSWe examined the interrelationship and functional significance of PCSK9 and cardiac myeloid cells in ischemic hearts from AMI-induced Pcsk9-/- and Lyz2crePcsk9fl/flmice, as well as in serum samples from coronary artery disease (CAD) patients treated with PCSK9 antibodies (Ab). Single-cell RNA sequencing (scRNA-seq) was conducted to identify heterogenous cardiac macrophage clusters and to investigate the impact of adaptive remodeling due to PCSK9 deficiency during AMI. Additionally, the regulatory effect of the myeloid-PCSK9/VEGF-C pathway was assessed in vitro as a potential therapeutic strategy. RESULTSOur study demonstrated that PCSK9 deficiency induces diverse changes in myeloid cells and macrophages, potentially offering cardiac protection following AMI, irrespective of LDL cholesterol homeostasis. The scRNA-seq identified a subset of PCSK9-dependent cardiac macrophages (PDCMs) enriched in activator protein-1 (AP-1)-related pathways, functioning as reparative macrophages. These PDCMs were shown to enhance vascular endothelial growth factor C (VEGF-C) secretion and activate Akt signaling in cardiac endothelial cells, leading to improved cardiac remodeling. Notably, CAD patients treated with PCSK9 inhibitors exhibited increased numbers of myeloid cells with PDCM-like features, including elevated VEGF-C levels, consistent with our findings in mice. COUNCLUSIONSTargeting PCSK9 in myeloid cells could offer cardioprotective effects by increasing AP-1 activity and VEGF-C expression of PDCMs, presenting a novel approach to preventing cardiac dysfunction in AMI. This strategy could expand the clinical use of existing PCSK9 inhibitors beyond just lowering LDL cholesterol. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIMyeloid-PCSK9 deficiency attenuated cardiac dysfunction post-acute myocardial infarction (AMI) without affecting plasma lipid levels. These findings position PCSK9 as a novel immune regulator of macrophages, revealing functions independent of its role in LDL cholesterol regulation. C_LIO_LIWe demonstrated PCSK9-dependent cardiac macrophages (PDCMs) that play a reparative role under ischemic conditions influenced by PCSK9, using single-cell RNA sequencing (scRNA-seq) of CD45+ leukocytes following AMI. C_LIO_LIStrong enrichment of AP-1 family proteins in PDCMs led to reparative VEGF-C signaling in endothelial cells and improved cardiac remodeling, independent of PCSK9s conventional role in cholesterol homeostasis. C_LIO_LIIn coronary artery disease (CAD) patients, PCSK9 inhibition augmented myeloid cell populations towards a reparative phenotype and elevated VEGF-C levels, aligning with our findings in mice. C_LI What Are the Clinical Implications?O_LIMyeloid-derived PCSK9 is pathobiologically significant, directly influencing immune functions and contributing to cardiac remodeling after AMI, suggesting that targeting myeloid-specific PCSK9 could be a valuable therapeutic approach. C_LIO_LIGiven that the reparative effects of PCSK9 inhibitors on macrophages are preserved in CAD patients, this strategy could broaden the clinical applications of existing PCSK9 inhibitors beyond LDL cholesterol regulation. C_LI

Pathological cardiac hypertrophy (PCH) is a precursor to heart failure, driven in part by dysregulated nuclear-localized cAMP (NLS-cAMP) signaling. Phosphodiesterase 3A2 (PDE3A2) is a key regulator of this nuclear cAMP microdomain, yet its selective modulation remains challenging. Here, we developed PDE3A2-modified RNA (PDE3A2-modRNA) to restore nuclear PDE3A2 levels and tested its efficacy in an angiotensin II-induced PCH model. Subcellular FRET imaging revealed that PCH hearts exhibit hyperactive NLS-cAMP due to PDE3A2 depletion. PDE3A2-modRNA selectively reduced NLS-cAMP without altering cytosolic cAMP, demonstrating precise microdomain regulation. In vivo, PDE3A2-modRNA improved cardiac function, attenuated hypertrophy and fibrosis, and shifted transcriptional programs toward physiological remodeling. Single-nucleus transcriptomics and immune profiling further revealed reduced oxidative stress and an improved cardiac microenvironment. These findings highlight PDE3A2-modRNA as a novel gene therapy that selectively restores nuclear cAMP homeostasis, reversing PCH-promoting transcriptional programs while preserving physiological cAMP signaling in cytosolic microdomains.

RationaleHypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. ObjectiveTo determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and ResultsWe evaluated the pan-KDM inhibitor JIB-04 in Myh6R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A- accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell- derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. ConclusionsPharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

Metabolic pathways, such as fatty acid oxidation and oxidative phosphorylation, can modulate inflammatory cells. However, little is known about the effects of the fatty acid synthesis pathway in macrophages on inflammation and cardiac remodeling after myocardial infarction (MI). Using spatial metabolomics, here we show that cardiac macrophages residing in the infarct synthesize de novo fatty acids and increase the production of fatty acid enzymes including ACLY and FASN. Mice deficient in myeloid Acly and Fasn have improved cardiac function after MI and reduced fibrosis. Combining Cleavage Under Targets and Release Using Nuclease (CUT&RUN), RNA sequencing analysis of Acly-/-macrophages, and macrophage-specific in vivo gene silencing, we demonstrate that ACLY acetylates the promoter region of the upstream regulator Krt17, which drives the production of pro-fibrotic cytokines, including IL-33. Single-cell RNA sequencing of cardiac fibroblasts shows that the expansion of a population of fibroblasts (Fibroblast 5) expressing high levels of extracellular matrix genes after MI is confined in the absence of macrophage Acly. Finally, the analysis of spatial multi-omics data of human hearts with MI uncovers myofibroblasts with the Fibroblast 5 gene signature. These myofibroblasts are located near cardiac macrophages expressing high levels of ACLY. In summary, we show that macrophage ACLY and FASN are deleterious in MI pathogenesis.

BackgroundMechanisms of macrophage switching from pro-inflammatory to anti-inflammatory phenotypes are not well understood. Circular RNAs (circRNAs), a new class of non-coding RNAs, are implicated in immune modulation. We recently identified circ-cdr1as as a regulator of macrophage phenotype in bone marrow derived macrophages (BMDM), however, their role in immunomodulation during cardiovascular injury remains unknown. MethodsCell-specific expression levels of circ-cdr1as was determined in a mouse hearts post-myocardial infarction (MI). Circ-cdr1as was overexpressed in fluorescently labeled BMDMs and injected into the ischemic myocardium immediately following MI. Effect of AAV9-mediated systemic delivery of circ-Cdr1as on post-MI cardiac function and structure was determined. Downstream mechanisms were studied using gain and loss of function strategies. ResultsCardiac cell specific expression analysis showed significant downregulation of circ-cdr1as only in macrophages and cardiomyocytes. Overexpression of circ-cdr1as in BMDMs, injected into the ischemic myocardium retained their anti-inflammatory phenotype and significantly improved left ventricular (LV) functions and reduced infarct size. Systemic delivery of AAV9-circ-cdr1as showed similar cardiac reparative activity. Mechanistically, circ-cdr1as directly binds and sponge microRNA-7 and increases the expression of target KLF4. Loss and gain of function studies show that modulation of miR-7 and KLF recapitulates macrophage phenotypic changes. ConclusionsCirc-cdr1as plays a crucial role in regulating the anti-inflammatory phenotype of macrophages through modulation of miR-7 and its target gene KLF4. Therefore, circ-cdr1as holds potential as an anti-inflammatory regulator in tissue inflammation post-cardiac injury. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIDespite continued research in elucidating mechanisms involved in cardiovascular disease and benefits of approved guideline-based therapies, the leading cause of deaths worldwide continues to be cardiovascular diseases (CVDs) with an increasing incidence of heart failure. C_LIO_LIAdvances in high-throughput RNA sequencing (RNA-seq) allowed the identification of novel transcripts such as microRNAs (miRNA), long non-coding RNAs (lncRNAs), and circular RNAs (circRNA). circular RNAs have recently emerged as promising candidates for targeted therapy due to their circular structure that confers resistance to exonucleases, their capability to regulate gene expression by modulating miRNA activity, sequester proteins by acting as protein sponges, C_LIO_LISeveral studies identified circRNAs to be differentially expressed following myocardial infarction (MI) and to play a role in immunity by contributing to the process of macrophage polarization, appropriate activation of macrophages when exposed to LPS, and inhibition of macrophage biogenesis. However, there are currently no published studies into the role of circular RNAs in the regulation of macrophage plasticity during cardiac injury. C_LI What New information Does This Article Contribute?O_LIWe provide evidence that circ-cdr1as expression is downregulated in the heart 3 days post MI and specifically in cardiomyocytes and macrophages. C_LIO_LIOur study provides promising evidence that overexpression of circ-cdr1as may be cardioprotective by reducing cardiomyocyte apoptosis, enhancing angiogenesis, limiting infarct size, increasing percentage of anti-inflammatory macrophages, and overall preserving post-MI cardiac function. C_LIO_LIMechanistically, we identified a reciprocal relationship between circ-cdr1as and miR-7 at 3 days post-MI and in naive, pro-, and anti-inflammatory macrophages indicating circ-cdr1as role as a miRNA sponge. This suggests that circ-cdr1as/miR-7/Klf4 play a crucial role in cardiac injury and macrophage phenotype. C_LI

AimsDoxorubicin (DOX) is a highly effective chemotherapeutic agent whose clinical use is limited by cumulative cardiotoxicity. The subcellular origins of early cardiac injury remain unclear but cardiomyocyte (CM) mitochondrial dysfunction is implicated. However, vulnerability of specific CM mitochondrial subpopulations is unknown. Building on our previous work linking the postnatal maturation of crest-associated subsarcolemmal mitochondria (SSM) at the CM surface to diastolic function, we investigated the spatial and temporal susceptibility of SSM during DOX exposure and their contribution to early diastolic dysfunction. Methods and resultsAdult male mice received chronic DOX treatment (5 mg/kg/week for 5 weeks) to mimic cumulative clinical exposure. Cardiac function was monitored longitudinally, during treatment and after protocol completion, using echocardiography-Doppler imaging, and global longitudinal strain (GLS). Subcellular mitochondrial remodeling was assessed using atomic force microscopy (AFM) and transmission electron microscopy (TEM). A tamoxifen-inducible, CM-specific Ephrin-B1 knockout model was used to probe the functional role of SSM in DOX-induced injury. DOX induced a progressive and selective loss of crest/SSM at the CM surface very early within 3 days of exposure, while the architecture of interfibrillar mitochondria IFM remained preserved. This early SSM depletion paralleled impaired myocardial relaxation reflected by a prolonged isovolumic relaxation time, along with reduced GLS, all preceding changes in left ventricular ejection fraction or detectable IFM abnormalities. Notably, in mice lacking Ephrin-B1, and therefore mature crest/SSM, DOX exposure triggered an unusually rapid onset of systolic dysfunction, highlighting the cardioprotective role of these surface mitochondrial populations. ConclusionsCrest/SSM at the CM surface are the earliest selective mitochondrial targets of DOX, and their loss precedes IFM remodeling. This spatial-temporal hierarchy reveals a compartment-specific functional distinction, with SSM supporting diastolic performance and IFM sustaining systolic contraction. Hence, preserving SSM emerges as a promising early target to prevent progression of anthracycline cardiotoxicity toward systolic failure. Clinically, our findings also support early diastolic monitoring as a sensitive approach for detecting anthracycline cardiotoxicity.

Myocardial infarction is a major cause of death worldwide. Effective treatments are required that limit adverse cardiac remodelling and preserve cardiac contractility following myocardial infarction, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile myocardium bordering a newly created infarct is functionally distinct from the remote surviving myocardium; it is also a major determinant of adverse cardiac remodelling and whole heart contractility. Expression of the transcription factor RUNX1 is increased in the border zone at 1 day after myocardial infarction, suggesting potential for targeted therapeutic intervention. Here we demonstrate that RUNX1 drives reductions in cardiomyocyte contractility, sarcoplasmic reticulum-mediated calcium release, mitochondrial density, and the expression of genes important for oxidative phosphorylation. Antagonising RUNX1 expression via short-hairpin RNA interference preserved cardiac contractile function following myocardial infarction when delivered either via direct adenoviral delivery into the border zone or via an adeno-associated virus vector administered intravenously. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with the essential co-factor CBF{beta}. Both tamoxifen-inducible Runx1-deficient and Cbf{beta}-deficient cardiomyocyte-specific mouse models demonstrated that antagonising RUNX1 function preserves the expression of genes important for oxidative phosphorylation following myocardial infarction. Our results confirm the translational potential of RUNX1 as a novel therapeutic target in myocardial infarction, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

BackgroundAmyloidosis is a major long-term complication of chronic disease; however, whether it represents one of the complications of post-myocardial infarction (MI) is yet to be fully understood. MethodsUsing wild-type and knocked-out MI mouse models and characterizing in vitro the exosomal communication between bone marrow-derived macrophages and activated mesenchymal stromal cells (MSC) isolated after MI, we investigated the mechanism behind Serum Amyloid A 3 (SAA3) protein overproduction in injured hearts. ResultsHere, we show that amyloidosis occurs after MI and that amyloid fibers are composed of macrophage-derived SAA3 monomers. SAA3 overproduction in macrophages is triggered by exosomal communication from a subset of activated MSC, which, in response to MI, acquire the expression of a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN). Cardiac MSCPDPN+ communicate with and activate macrophages through their extracellular vesicles or exosomes. Specifically, MSCPDPN+ derived exosomes (MSCPDPN+ Exosomes) are enriched in SAA3 and exosomal SAA3 protein engages with Toll-like receptor 2 (TRL2) on macrophages, triggering an overproduction and impaired clearance of SAA3 proteins, resulting in aggregation of SAA3 monomers as rigid amyloid deposits in the extracellular space. The onset of amyloid fibers deposition alongside extra-cellular-matrix (ECM) proteins in the ischemic heart exacerbates the rigidity and stiffness of the scar, hindering the contractility of viable myocardium and overall impairing organ function. Using SAA3 and TLR2 deficient mouse models, we show that SAA3 delivered by MSCPDPN+ exosomes promotes post-MI amyloidosis. Inhibition of SAA3 aggregation via administration of a retro-inverso D-peptide, specifically designed to bind SAA3 monomers, prevents the deposition of SAA3 amyloid fibrils, positively modulates the scar formation, and improves heart function post-MI. ConclusionOverall, our findings provide mechanistic insights into post-MI amyloidosis and suggest that SAA3 may be an attractive target for effective scar reversal after ischemic injury and a potential target in multiple diseases characterized by a similar pattern of inflammation and amyloid deposition. NOVELTY AND SIGNIFICANCEWhat is known? O_LIAccumulation of rigid amyloid structures in the left ventricular wall impairs ventricle contractility. C_LIO_LIAfter myocardial infarction cardiac Mesenchymal Stromal Cells (MSC) acquire Podoplanin (PDPN) to better interact with immune cells. C_LIO_LIAmyloid structures can accumulate in the heart after chronic inflammatory conditions. C_LI What information does this article contribute? O_LIWhether accumulation of cumbersome amyloid structures in the ischemic scar impairs left ventricle contractility, and scar reversal after myocardial infarction (MI) has never been investigated. C_LIO_LIThe pathophysiological relevance of PDPN acquirement by MSC and the functional role of their secreted exosomes in the context of post-MI cardiac remodeling has not been investigated. C_LIO_LIAmyloid structures are present in the scar after ischemia and are composed of macrophage-derived Serum Amyloid A (SAA) 3 monomers, although mechanisms of SAA3 overproduction is not established. C_LI SUMMARY OF NOVELTY AND SIGNIFICANCEHere, we report that amyloidosis, a secondary phenomenon of an already preexisting and prolonged chronic inflammatory condition, occurs after MI and that amyloid structures are composed of macrophage-derived SAA3 monomers. Frequently studied cardiac amyloidosis are caused by aggregation of immunoglobulin light chains, transthyretin, fibrinogen, and apolipoprotein in a healthy heart as a consequence of systemic chronic inflammation leading to congestive heart failure with various types of arrhythmias and tissue stiffness. Although chronic MI is considered a systemic inflammatory condition, studies regarding the possible accumulation of amyloidogenic proteins after MI and the mechanisms involved in that process are yet to be reported. Here, we show that SAA3 overproduction in macrophages is triggered in a Toll-like Receptor 2 (TLR2)-p38MAP Kinase-dependent manner by exosomal communication from a subset of activated MSC, which, in response to MI, express a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin. We provide the full mechanism of this phenomenon in murine models and confirm SAA3 amyloidosis in failing human heart samples. Moreover, we developed a retro-inverso D-peptide therapeutic approach, "DRI-R5S," specifically designed to bind SAA3 monomers and prevent post-MI aggregation and deposition of SAA3 amyloid fibrils without interfering with the innate immune response.