Regulation of energy metabolism is pivotal in the development of cardiovascular diseases. Dysregulation in mitochondrial fatty acid oxidation has been linked to cardiac lipid accumulation and diabetic cardiomyopathy. Sirtuin 1 (SIRT1) is a deacetylase that regulates the acetylation of various proteins involved in mitochondrial energy metabolism. SIRT1 mediates energy metabolism by directly and indirectly affecting multiple aspects of mitochondrial processes, such as mitochondrial biogenesis. SIRT1 interacts with essential mitochondrial energy regulators such as peroxisome proliferator-activated receptor-α (PPARα), PPARγ coactivator-1α, estrogen-related receptor-α, and their downstream targets. Apart from that, SIRT1 regulates additional proteins, including forkhead box protein O1 and AMP-activated protein kinase in cardiac disease. Interestingly, studies have also shown that the expression of SIRT1 plays a dual-edged role in energy metabolism. Depending on the physiological state, SIRT1 expression can be detrimental or protective. This review focuses on the molecular pathways through which SIRT1 regulates energy metabolism in cardiovascular diseases. We will review SIRT1 and discuss its role in cardiac energy metabolism and its benefits and detrimental effects in heart disease.

Cardiovascular disease (CVD) is a leading cause of death worldwide, and the number of patients with CVD continues to increase despite extensive research and developments in this field. Chronic inflammation is a pivotal pathological component of CVD, and unveiling new proinflammatory factors will help devise novel preventive and therapeutic strategies. The extracellular matrix (ECM) not only provides structural support between cells but also contributes to cellular functions. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein that is particularly induced during development and tissue remodeling. A proinflammatory role for SPARC has been demonstrated in various animal models, such as in the lipopolysaccharide-induced footpad model and dextran sodium sulfate-induced colitis model. Recent clinical studies reported a positive correlation between elevated plasma SPARC levels and hypertension, obesity, and the inflammatory marker high-sensitive C-reactive protein. In addition, SPARC gene deletion attenuates the cardiac injury induced by aging, myocardial infarction, and pressure load, suggesting that SPARC has deleterious effects on CVD. This review summarizes the regulatory and proinflammatory mechanisms of SPARC on CVD, chronic kidney disease (CKD), and cerebrovascular disease and discusses the rationale behind measuring SPARC as a biomarker of CVD and the effects of inhibition of SPARC in the prevention and treatment of CVD.

Thyroid hormone dysfunction is frequently observed in patients with chronic illnesses including heart failure, which increases the risk of adverse events. This study examined the effects of thyroid hormones (THs) on cardiac transverse-tubule (TT) integrity, Ca²⁺ sparks, and nanoscale organization of ion channels in excitation-contraction (EC) coupling, including L-type calcium channel (Ca_V1.2), ryanodine receptor type 2 (RyR2), and junctophilin-2 (Jph2). TH deficiency was established in adult female rats by propyl-thiouracil (PTU) ingestion for 8 wk; followed by randomization to continued PTU without or with oral triiodo-l-thyronine (T3; 10 µg/kg/day) for an additional 2 wk (PTU + T3). Confocal microscopy of isolated cardiomyocytes (CMs) showed significant misalignment of TTs and increased Ca²⁺ sparks in thyroid-deficient CMs. Density-based spatial clustering of applications with noise (DBSCAN) analysis of stochastic optical reconstruction microscopy (STORM) images showed decreased (P < 0.0001) RyR2 cluster number per cell area in PTU CMs compared with euthyroid (EU) control myocytes, and this was normalized by T3 treatment. Ca_V1.2 channels and Jph2 localized within a 210 nm radius of the RyR2 clusters were significantly reduced in PTU myocytes, and these values were increased with T3 treatment. A significant percentage of the RyR2 clusters in the PTU myocytes had neither Ca_V1.2 nor Jph2, suggesting fewer functional clusters in EC coupling. Nearest neighbor distances between RyR2 clusters were greater (P < 0.001) in PTU cells compared with EU- and T3-treated CMs that correspond to disarray of TTs at the sarcomere z-discs. These results support a regulatory role of T3 in the nanoscale organization of RyR2 clusters and colocalization of Ca_V1.2 and Jph2 in optimizing EC coupling.NEW & NOTEWORTHY Thyroid hormone (TH) dysfunction exacerbates preexisting heart conditions leading to an increased risk of premature morbidity/mortality. Triiodo-l-thyronine (T3) optimizes cardiac excitation-contraction (EC) coupling by maintaining myocardial T-tubule (TT) structures and organization of calcium ion channels. Single-molecule localization microscopy shows T3 effects on the clustering of ryanodine receptors (RyR2) with colocalization of L-type calcium channels (Ca_V1.2) and junctophilin-2 (Jph2) at TT-SR structures. Heart disease with subclinical hypothyroidism/low T3 syndrome may benefit from TH treatment.

Systemic inflammation is reported in normal-weight obesity (NWO) and metabolically healthy obesity (MHO), which may be linked to their increased cardiovascular disease (CVD) risk. Yet, drivers of this inflammation remain unclear. We characterized factors known to influence inflammatory status (i.e., intestinal permeability, adipose tissue, diet quality, microbiota), and their relationships with measured inflammation, in NWO and MHO, healthy control subjects (CON), and metabolically unhealthy obesity (MUO; N = 80; n = 20/group). Serum indicators of intestinal permeability and inflammation were assessed by ELISA and/or multiplex. Total, visceral, and percent body fat were measured with dual-energy X-ray absorptiometry (DXA). Fecal microbiota composition was assessed via 16S rRNA sequencing (n = 9–10/group). For C-reactive protein (CRP), MUO > NWO > CON (P < 0.0001). In MHO, CRP was intermediate and similar to both MUO and NWO. Lipopolysaccharide binding protein (LBP) and the ratio of LBP to soluble CD14 (sCD14) were higher in MHO and MUO vs. CON/NWO (P < 0.0001). Across correlation and regression analyses, LBP consistently displayed the strongest relationships with CRP in the entire sample (r = 0.78; β = 0.57; P < 0.0001) and in MHO (r = 0.74; P < 0.01) but not NWO (r = 0.37; P = 0.11). Shannon index was higher in CON compared with MUO (P < 0.05) and inversely correlated with CRP in the full sample (r = −0.37; P < 0.05). These data are consistent with the notion that intestinal permeability is associated with low-grade inflammation in MHO, which could be implicated in this population’s reported CVD risk.NEW & NOTEWORTHY This is the first study to our knowledge to examine biomarkers of intestinal permeability in normal-weight obesity and one of few assessing microbiota compositions in this population. Additionally, we report that individuals with metabolically healthy obesity and metabolically unhealthy obesity displayed similar evidence of intestinal permeability, which was more strongly associated with systemic inflammation than total and visceral adipose tissue mass.

Heart failure is a major cause of mortality following myocardial infarction. Neutrophils are among the first immune cells to accumulate in the infarcted region. Although beneficial functions of neutrophils in heart injury are now appreciated, neutrophils are also well known for their ability to exacerbate inflammation and promote tissue damage. Myocardial infarction induces hypoxia, where hypoxia-inducible factors (HIFs) are activated and play critical roles in cellular functions. In this context, the role of Hif2α in neutrophils during myocardial infarction is unknown. Here, we demonstrate that neutrophil Hif2α deletion markedly attenuates myocardial infarct size, improves cardiac function, reduces neutrophil survival and tissue accumulation, and correlates with increased macrophage engulfment rates. Mechanistic studies revealed that Hif2α promotes neutrophil survival through binding to hypoxia response element (HRE) in the promoter region of Birc2 to regulate expression of the prosurvival factor, cellular inhibitor of apoptosis protein-1 (cIAP1). Inhibition of cIAP1 in neutrophils using the pharmacological agent, Birinapant resulted in increased cell death, establishing a critical role of cIAP1 downstream of Hif2α in neutrophil survival. Taken together, our data demonstrate a protective effect of Hif2α deletion in neutrophils on cardiac injury outcomes through modulation of neutrophil cell survival.NEW & NOTEWORTHY Hif2α in neutrophils increases infarct size, cardiac dysfunction, and ventricular scar after myocardial infarction. Hif2α in neutrophils supports neutrophil survival via cIAP-1 signaling and delays macrophage engulfment.

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well characterized. To define whether the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild-type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4 wk of TAC compared with WT cohorts. Both USP20-KO TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume overload states and decompensated heart failure. In contrast, WT-TAC mice displayed an increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis, and mouse mortality were augmented in USP20-KO-TAC compared with WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling, was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts, indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.NEW & NOTEWORTHY We identify ubiquitin-specific peptidase 20 (USP20) as an important enzyme that is required for cardiac homeostasis and function, particularly during myocardial pressure overload. USP20 regulates protein stability of cardiac MYH7, an essential molecular motor protein expressed in sarcomeres; loss-of-function mutations of MYH7 are associated with human hypertrophic cardiomyopathy, cardiac failure, and sudden death. Enhancing USP20 activity could be a potential therapeutic approach to prevent the development of maladaptive state of eccentric hypertrophy and heart failure.

Left ventricular pressure overload (LVPO) can lead to heart failure with a preserved ejection fraction (HFpEF) and LV chamber stiffness (LV K_c) is a hallmark. This project tested the hypothesis that the development of HFpEF due to an LVPO stimulus will alter posttranscriptional regulation, specifically microRNAs (miRs). LVPO was induced in pigs (n = 9) by sequential ascending aortic cuff and age- and weight-matched pigs (n = 6) served as controls. LV function was measured by echocardiography and LV K_c by speckle tracking. LV myocardial miRs were quantified using an 84-miR array. Treadmill testing and natriuretic peptide-A (NPPA) mRNA levels in controls and LVPO were performed (n = 10, n = 9, respectively). LV samples from LVPO and controls (n = 6, respectively) were subjected to RNA sequencing. LV mass and K_c increased by over 40% with LVPO (P < 0.05). A total of 30 miRs shifted with LVPO of which 11 miRs correlated to LV K_c (P < 0.05) that mapped to functional domains relevant to K_c such as fibrosis and calcium handling. LVPO resulted in reduced exercise tolerance (oxygen saturation, respiratory effort) and NPPA mRNA levels increased by fourfold (P < 0.05). RNA analysis identified several genes that mapped to specific miRs that were altered with LVPO. In conclusion, a specific set of miRs are changed in a large animal model consistent with the HFpEF phenotype, were related to LV stiffness properties, and several miRs mapped to molecular pathways that may hold relevance in terms of prognosis and therapeutic targets.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is an ever-growing cause for the HF burden. HFpEF is particularly difficult to treat as the mechanisms responsible for this specific form of HF are poorly understood. Using a relevant large animal model, this study uncovered a unique molecular signature with the development of HFpEF that regulates specific biological pathways relevant to the progression of this ever-growing cause of HF.

The Ras-related GTP-binding protein D (RRAGD) gene plays a crucial role in cellular processes. Recently, RRAGD variants found in patients have been implicated in a novel disorder with kidney tubulopathy and dilated cardiomyopathy. Currently, the consequences of RRAGD variants at the organismal level are unknown. Therefore, this study investigated the impact of RRAGD variants on cardiac function using a zebrafish embryo model. Furthermore, the potential usage of rapamycin, an mTOR inhibitor, as a therapy was assessed in this model. Zebrafish embryos were injected with RRAGD p.S76L and p.P119R cRNA and the resulting heart phenotypes were studied. Our findings reveal that overexpression of RRAGD mutants resulted in decreased ventricular fractional shortening, ejection fraction, and pericardial swelling. In RRAGD S76L-injected embryos, lower survival and heartbeat were observed, whereas survival was unaffected in RRAGD P119R embryos. These observations were reversible following therapy with the mTOR inhibitor rapamycin. Moreover, no effects on electrolyte homeostasis were observed. Together, these findings indicate a crucial role of RRAGD in cardiac function. In the future, the molecular mechanisms by which RRAGD variants result in cardiac dysfunction and if the effects of rapamycin are specific for RRAGD-dependent cardiomyopathy should be studied in clinical studies.NEW & NOTEWORTHY The resultant heart-associated phenotypes in the zebrafish embryos of this study serve as a valuable experimental model for this rare cardiomyopathy. Moreover, the potential therapeutic property of rapamycin in cardiac dysfunctions was highlighted, making this study a pivotal step toward prospective clinical applications.

Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell-cycle progression and proliferation compared with WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared with WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathological phenotypic switching and proliferation in VSM.NEW & NOTEWORTHY Extracellular acidosis remains an understudied feature of many pathologies. We examined a potential regulatory role for pH-sensing GPR68 in vascular smooth muscle (VSM) growth in the context of CVD. With in vivo and in vitro growth models with GPR68-deficient mice and GPR68 induction strategies, novel findings revealed capacity of GPR68 to attenuate growth through the small GTPase Rap1A. These observations highlight GPR68 and its effector Rap1A as possible therapeutic targets to combat pathological VSM growth.

Vascular dysfunction, marked by lower endothelial function and increased aortic stiffness, is a nontraditional risk factor that precedes the development of cardiovascular disease (CVD). However, the age at which these changes in vascular function occur in women and the degree to which reproductive hormones mediate these changes has not been characterized. Women free from major disease were enrolled across the adult life span (aged 18–70 yr, n = 140). Endothelial function was assessed as flow-mediated dilation (FMD) of the brachial artery during reactive hyperemia using duplex ultrasound and expressed as percent dilation. Aortic stiffness was measured by carotid-femoral pulse wave velocity (cfPWV). Blood samples were obtained to quantify reproductive hormone concentration. Regression models determined age-related breakpoints and mediating factors between age and vascular outcomes. FMD declined with age with a breakpoint and steeper decline occurring at 47 yr of age. Thereafter, age was independently associated with lower FMD (B = −0.13, P < 0.001). cfPWV was relatively stable until a breakpoint at age 48, and age was independently associated with higher cfPWV thereafter (B = 0.10, P < 0.001). Path analysis revealed that the association between age and FMD was partially mediated by follicle-stimulating hormone (ab_ind = 0.051, P = 0.01) and progesterone (ab_ind = 0.513, P < 0.001) but not estradiol (ab_ind = −0.004, P = 0.08). No mediation was present for cfPWV. Age was associated with endothelial dysfunction and aortic stiffness in women beginning at 47 and 48 yr old, respectively, 3 to 4 yr before the average age of menopause. The association between age and endothelial dysfunction was explained in part by elevations in follicle-stimulating hormone and progesterone, but not declining estradiol.NEW & NOTEWORTHY We demonstrate that the age at which endothelial function declines and aortic stiffness increases in healthy women is 47 and 48, respectively. The inflection point in flow-mediated dilation (FMD) is 6 yr earlier than previously reported, and the association between age and FMD was mediated by follicle-stimulating hormone (FSH) and progesterone (P₄) but not estradiol (E₂).

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca²⁺ handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24 h or 7 days at a physiological rate of 60 beats/min on cellular electrophysiology and Ca²⁺ cycling in late-stage, differentiated hiPSC-CM (>90% troponin⁺, >60 days postdifferentiation). Electric culture pacing for 7 days did not influence cardiomyocyte cell size, apoptosis, or generation of reactive oxygen species in differentiated hiPSC-CM compared with 24-h pacing. However, epifluorescence measurements revealed that electric pacing for 7 days improved systolic Ca²⁺ transient amplitude and Ca²⁺ transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca²⁺ load and SERCA activity. Diastolic Ca²⁺ leak was not changed in line-scanning confocal microscopy, suggesting that the improvement in systolic Ca²⁺ release was not associated with a higher open probability of ryanodine receptor (RyR)2 during diastole. Whereas bulk cytosolic Na⁺ concentration and Na⁺/Ca²⁺ exchanger (NCX) activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole cell voltage-clamp measurements that chronic pacing for 7 days led to a decrease in late Na⁺ current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca²⁺ handling and modulates the electrophysiology of late-stage, differentiated hiPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.NEW & NOTEWORTHY Electric pacing is increasingly used in research with human induced pluripotent stem cell cardiomyocytes (hiPSC-CM), for example to simulate arrhythmias but also to enhance maturity. Therefore, it is mandatory to understand the effects of pacing itself on cellular electrophysiology in late-stage, matured hiPSC-CM. This study provides an electrophysiological characterization of the effects of chronic electric pacing at a physiological rate on differentiated hiPSC-CM.

The alpha 7 nicotinic acetylcholine receptor (α7nAChR) regulates inflammation in experimental models and is expressed in human peripheral blood mononuclear cells (PBMCs) and in human atherosclerotic plaques. However, its role in regulating inflammation in patients with cardiovascular disease is unknown. This study aims to investigate whether α7nAChR stimulation can reduce the inflammatory response in PBMCs from patients with newly diagnosed coronary artery disease (CAD). Human PBMCs, extracted from patients with verified CAD (n = 38) and control participants with healthy vessels (n = 38), were challenged in vitro with lipopolysaccharide (LPS) in combination with the α7nAChR agonist PHA 568487. Cytokine levels of the supernatants were analyzed using a multiplex immunoassay. Patients in the CAD group were reexamined after 6 mo. The immune response to LPS did not differ between PBMCs from control and CAD groups. α7nAChR stimulation decreased TNFα in both control and CAD groups. The most pronounced effect of α7nAChR stimulation was observed in patients with CAD at their first visit, where 15 of 17 cytokines were decreased [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-17A, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, and TNFα]. In conclusion, stimulation with α7nAChR agonist PHA 568487 dampens the inflammatory response in human PBMCs. This finding suggests that the anti-inflammatory properties of the α7nAChR may have a role in treating CAD.NEW & NOTEWORTHY The α7nAChR is an important regulator of inflammation; however, its anti-inflammatory function in patients with newly diagnosed coronary artery disease (CAD) remains unclear. We demonstrate that stimulation of α7nAChR with PHA 568487 attenuates the inflammatory response in immune cells extracted from healthy controls and patients with newly diagnosed CAD, with a more pronounced effect observed in patients with CAD. This suggests that the anti-inflammatory properties of α7nAChR may have a role in treating chronic inflammatory diseases.

Mitochondrial dysfunction contributes significantly to the development of atrial fibrillation (AF). Conflicting data regarding the atrial pacing and the risk of AF existed, and the impact of atrial pacing on mitochondrial function remains unknown. Therefore, we sought to examine the association between atrial pacing percentage and mitochondrial function in patients with cardiovascular implantable electronic devices (CIEDs) with atrial pacing capability. This is a cross-sectional study involving 183 patients with CIEDs with atrial pacing capability. The oxidative stress and mitochondrial function were determined in peripheral blood mononuclear cells (PBMCs). Among 183 patients, 55.7% had permanent pacemakers, 7.7% had defibrillators, and 36.6% had cardiac resynchronization therapy. Mean age was 67.5 ± 14.7 yr with 51% being male. Mean left ventricular ejection fraction (LVEF) was 53.9 ± 16.8%. We demonstrated that the presence of atrial pacing above 50% correlated with higher levels of mitochondrial spared respiratory capacity (P = 0.043) and coupling efficiency (P = 0.045). After adjusting with multiple linear regression for age, sex, LVEF, history of AF, sick sinus syndrome, comorbidities, estimated glomerular filtration rate (eGFR), cardiac resynchronization therapy (CRT), and percentage of ventricular pacing, our findings revealed a statistically significant association between a higher percentage of atrial pacing and increased spared respiratory capacity (β, 0.217, P = 0.046), lower nonmitochondrial respiration (β, −0.230; P = 0.023), and proton leak (β, −0.247; P = 0.022). We demonstrated that atrial pacing enhances mitochondrial performance in PBMCs and left ventricular contractile performance in patients with CIEDs. This observation may serve as an additional support for the preventive effect of atrial pacing in the prevention of atrial arrhythmia.NEW & NOTEWORTHY Atrial pacing enhances mitochondrial spare respiratory capacity and reduces proton leak. This finding may provide further evidence supporting the preventive role of atrial pacing in reducing the risk of atrial fibrillation in patients with cardiac implantable electronic devices.

Lymphatic vessels of the heart undergo dynamic remodeling in response to physiological and pathological cardiovascular events such as development, adult cardiac maintenance, and injury repair. During pregnancy, the cardiovascular system undergoes physiological changes to meet the increased demand for blood supply to the fetus. These extensive physiological changes make pregnancy and delivery a high-risk period in a woman’s life. However, whether and how cardiac lymphatics change during pregnancy is largely undefined. Therefore, we used whole mount immunofluorescent labeling and quantitative morphometric analysis to characterize the changes in cardiac lymphatic vasculature during pregnancy using two genetically distinct inbred mouse strains, C57BL/6J and BALB/cJ. When compared with age-matched, nonpregnant C57BL/6J control mice, the hearts of C57BL/6J dams in late pregnancy [gestation day 17.5 (G17.5)] undergo physiological hypertrophy. However, there were no significant changes in the cardiac lymphatic vasculature. In contrast, BALB/cJ mice do not exhibit pregnancy-induced cardiac hypertrophy at G17.5 compared with age-matched, nonpregnant mice. Yet interestingly, the cardiac lymphatic vasculature of pregnant BALB/cJ dams undergoes extensive morphological changes, including decreased lymphatic length, number of end points, and vessel branch-point junctions on the ventral side of the heart. These findings underscore the complexity of genetic and physiological factors that contribute to the heterotypic remodeling of cardiac lymphatics during late pregnancy.NEW & NOTEWORTHY Cardiac lymphatics remodel in response to physiological and pathological stresses. This study is the first to investigate cardiac lymphatic vessel changes during pregnancy. BALB/cJ mice, which do not undergo pregnancy-induced cardiac hypertrophy, show decreased lymphatic length, number of end points, and junctions on the ventral side during pregnancy. In contrast, C57BL/6J mice, which undergo pregnancy-induced cardiac hypertrophy, had no such changes. These findings underscore the complexity of genetic and physiological factors contributing to cardiac lymphatic remodeling.

microRNAs (miRNAs) have been intensively studied as valuable biomarkers in cardiometabolic disease. Typically, miRNAs are detected in plasma or serum, but the use of samples collected in heparinized tubes is problematic for miRNA studies using quantitative PCR (qPCR). Heparin and its derivatives interfere with qPCR-based analysis, leading to a substantial reduction or even complete loss of detectable miRNA levels. Given that red blood cells (RBCs) express abundant miRNAs, whose expression is altered in cardiometabolic disease, RBCs could serve as an attractive alternative in biomarker studies. Here, we aim to explore the stability of miRNAs in RBCs collected from whole blood with different anticoagulants and thereby the potential of RBCs as alternative materials for miRNA biomarker studies. miRNA profiling was performed in human RBCs via RNA sequencing, followed by qPCR validation of selected miRNAs in RBCs and plasma in both heparinized and EDTA tubes. RNA sequencing revealed abundant miRNA presence in RBCs isolated from blood collected in EDTA tubes. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected at comparable levels in RBCs isolated from both heparinized and EDTA tubes but not in plasma from heparinized tubes. Of note, miR-210-3p levels were consistently lower in RBCs from individuals with type 2 diabetes compared with healthy controls, regardless of anticoagulant type, supporting their potential as biomarker materials. In conclusion, RBCs offer a promising alternative for miRNA biomarker studies, overcoming heparin-related challenges.NEW & NOTEWORTHY microRNAs are valuable biomarkers in cardiometabolic disease, but heparinized tubes hinder their detection because of qPCR interference. RBCs, which express abundant microRNAs like miR-210-3p, may serve as an alternative. microRNAs, including miR-210-3p, are consistently detectable in RBCs at comparable levels between heparinized and EDTA tubes. miR-210-3p levels in RBCs are similarly reduced in heparinized tubes of patients with type 2 diabetes. Thus, RBCs offer a promising solution for miRNA biomarker studies, overcoming heparin-related challenges.