Background/Objectives: The non-neuronal cardiac cholinergic system (NNCCS) is known to synthesize ACh independently of the parasympathetic nervous system, thereby regulating cardiac homeostasis, which includes sustainability of energy metabolism, anti-inflammatory and anti-ischemic properties, electrical stability, and mitochondrial calcium handling. Given these beneficial functions of NNCCS, we were prompted to search for an inducer. One such inducer is SNPiP, a novel low-molecular-weight chemical compound developed by us. SNPiP accelerates ACh synthesis in the heart via cGMP elevation and, intriguingly, enhances diastolic function, increasing cardiac output and end-systolic pressure without elevating heart rate. However, the pharmacokinetics of SNPiP remain unknown, which led us to conduct the present study. Methods and Results: We found that the half-life of SNPiP in the blood was extremely short, similar to that of a nitric oxide (NO) donor, S-nitroso-N-acetyl-DL-penicillamine. This short half-life is caused by the rapid distribution of SNPiP into organs, including the heart, kidney, and liver. In addition, once transferred into blood cells, SNPiP itself became stable and remained intact for up to 1 h. Moreover, the short half-life was partly explained by the rapid degradation of SNPiP and concomitant loss of the nitroso group in the blood. Notably, when rats were treated with SNPiP, NO levels in the heart elevated bimodally: immediately after administration and again about 12 h later, coinciding with the previous report of NNCCS upregulation and accelerated ACh synthesis with NO production. Importantly, our previous transcriptome analysis of SNPiP-treated hearts supports these findings, as it revealed upregulation of diastolic function-related genes and proteins. Conclusions: Collectively, these results clarify the pharmacokinetics of SNPiP and demonstrate that, despite a shorter half-life, SNPiP is efficiently distributed to the heart, where it confers beneficial effects through induction of NNCCS.

The Developmental Origins of Health and Disease (DOHaD) hypothesis highlights the pivotal role of early-life nutrition in shaping lifelong health and disease risk. Low birth weight (LBW) remains a major public health issue associated with increased susceptibility to metabolic and cardiovascular disease, underscoring the need for early nutritional interventions. We investigated whether dietary supplementation with soy protein isolate (SPI) during lactation could mitigate adverse developmental programming in a rat model of LBW induced by maternal calorie restriction. Dams received an SPI-supplemented diet during lactation, and offspring were evaluated for postnatal growth, circulating IGF-1 and corticosterone concentrations, and pituitary expression of Gas5 lncRNA, miR-23b, and Pomc. Stress responsiveness and glucocorticoid receptor sensitivity were also assessed. SPI supplementation restored postnatal growth and IGF-1 concentrations in female offspring, and in males, it normalized pituitary Gas5 lncRNA and Pomc mRNA expressions, reduced stress-induced corticosterone hypersecretion, and improved pituitary glucocorticoid sensitivity. These findings indicate that SPI intervention during lactation can partially reverse epigenetic dysregulation of the stress and somatotropic axes caused by fetal undernutrition. Nutritional modulation during lactation thus represents a critical window for early intervention in LBW offspring. SPI supplementation may enhance endocrine and metabolic resilience, providing a practical nutritional programming approach to reduce future disease risk, consistent with the DOHaD paradigm.

Our previous studies, as well as other investigations, demonstrated that non-neuronal acetylcholine (ACh) produced by cardiomyocytes-that is, the non-neuronal cardiac cholinergic system (NNCCS)-is indispen- sable for sustaining the physiological functions and structural integrity of cardiomyocytes and for protect- ing the heart from ischemic/hypoxic insults, hypertrophic stress, and hypersympathetic conditions. These findings were supported by pharmacologically manipulated models in non-neuronal ACh systems and by gain-or loss-of-function models in the NNCCS. Nevertheless, the mechanisms underlying this phenomenon (i.e., sustention and protection) and the target of the NNCCS in cardiomyocytes remain to be fully elucidated. Our conditional murine model with heart-specific deletion of the choline acetyltransferase (ChAT) gene in the heart (hChAT KO mice) revealed cardiac dysfunction associated with heart failure symptoms. The representative culprit targets were the mitochondria with a disorganized appearance and dysfunction, accompanied by a reduction in mitochondrial DNA, membrane potential, and ATP production. Alternatively, malfunctioning mitochondria impaired cardiac energy metabolism and nicotinic receptor-mediated calcium responses in the mitochondria and down-regulated the mitochondrial calcium uniporter (MCU), leading to poor calcium handling by the mitochondria. The impaired cardiac function in hChAT KO mice induced systemic inflammatory responses and attenuated blood-brain barrier function, further influencing higher brain functions, including the aggravation of depression-like phenomenon. These specifically characteristic phenotypes indicate that the NNCCS principally plays a crucial role in sustaining mitochondrial functions through nicotinic receptors in the mitochondria and that the signal is indispensable for maintaining mitochondrial functions and integrity.

Isolation of primary cardiomyocytes can be performed for studies from fetuses and neonates. Compared with rat neonatal cardiomyocytes, mouse neonatal cardiomyocytes are sensitive to enzyme digestion. Therefore, they need to optimize a concentration of collagenase and a time point to collect the heart after birth. In addition, neonatal cardiomyocytes have unique characteristics to be recognized although the preparation steps are not complicated. Therefore, depending on a researcher's interest and aim, they consider which sources of cardiomyocytes are suitable and convenient, for example, a rat or mouse, as well as a fetus and neonate.

Polyacrylamide gel electrophoresis (PAGE) has traditionally been used to analyze lipoproteins in human and animal sera. Most conventional PAGE systems for lipoprotein analysis use disc electrophoresis, which limits the number of samples simultaneously analyzed and does not allow the comparison of electrophoretic profiles of multiple samples under the same conditions. Other methods, such as high performance liquid chromatography and agarose gel electrophoresis, are also used for lipoprotein analysis; however, they require specialized equipment and expertise. Here, we report an improved slab PAGE method with high-throughput, cost-effective, simple, rapid, and reproducible analysis of lipoproteins in the human serum. This system uses Tris-histidine as the running buffer and imidazole buffer for the polyacrylamide gel; therefore we named it the histidine-imidazole polyacrylamide gel electrophoresis (HI-PAGE) system. HI-PAGE effectively prevented band distortions of lipoproteins within an hour of electrophoresis and guaranteed precise quantitative analyses of multiple samples containing lipid-protein complexes. Furthermore, we pre-stained the lipoproteins with a fluorescent dye, Nile Red, and applied it to HI-PAGE electrophoresis. Fluorescent HI-PAGE (fHI-PAGE) was applied to the clinical samples and revealed that the method is highly sensitive and allows the quantitative detection of lipoproteins in human serum. ### Competing Interest Statement The authors have declared no competing interest.

Background: Polyacrylamide gel electrophoresis (PAGE) has long been used for lipoprotein analysis, enabling the separation and profiling of lipoprotein fractions such as LDL and HDL. However, conventional disc PAGE systems are limited by low throughput and inability to directly compare multiple samples under identical conditions. Alternative methods, including high-performance liquid chromatography and agarose gel electrophoresis, require specialized equipment and expertise, limiting their clinical utility. Methods: We present a colorimetric and fluorescence-based histidine-imidazole PAGE (HI-PAGE) system that provides rapid, cost-effective, and reproducible separation and profiling of lipoproteins in human serum. By combining electrophoretic separation with lipid-specific fluorescent staining using Nile Red, the fluorescence-based HI-PAGE (fHI-PAGE) not only visualizes distinct migration patterns of lipoprotein fractions, but also enables the quantification of LDL-cholesterol (LDL-C). Clear resolution of LDL and other lipoprotein fractions was achieved within 1 h without band distortion, allowing for direct comparison of multiple samples on a single gel. Results: We validated fHI-PAGE using serum from healthy individuals and patients, demonstrating that its fluorescence-based detection was more sensitive than conventional Sudan Black B staining while providing LDL-C estimates concordant with values calculated by the Friedewald formula. Moreover, fHI-PAGE proved advantageous in cases of hypertriglyceridemia, where Friedewald calculations are unreliable. Conclusions: These findings establish fHI-PAGE as a practical and clinically applicable platform for simultaneous lipoprotein profiling and LDL-C quantification.

Corticotropin-releasing factor (CRF) plays roles in stress-related responses through its type 1 (CRF1) and type 2 receptors. Both CRF and CRF1 are expressed in the rat colon. Peripheral CRF administration and various stressors increase colonic motility and defecation. Stress induces CRF release in the colon, suggesting CRF may mediate stress-related responses of the colon. The vagal nodose ganglion (NG) transduces visceral information, including colonic sensation, to the brain. However, it remains unclear whether the CRF/CRF1 system is involved in vagal afferent functions. This study, therefore, aimed to clarify the involvement of the CRF/CRF1 system in relaying visceral sensory information to the brain and the effect of stress exposure on vagal nerve function. The experiments were conducted in male rats. First, CRF1-like immunoreactivity (CRF1-LI) was characterized in the NG. Second, the effects of vagotomy on CRF1-LI in the NG, intraperitoneally administered CRF-induced fecal output, and c-Fos expression in the nucleus tractus solitarius (NTS) were evaluated. Subsequently, a fast blue retrograde tracer was microinjected into the proximal colon. Finally, we analyzed CRF- or stress-induced phosphorylation of cyclic AMP-response element-binding protein (pCREB) in the NG. CRF1 mRNA and CRF1-LI were detected, and CRF1-LI accumulated on the proximal side of the ligated region of the nerve trunk, and CRF1-LI was detected in most cholinergic neurons. CRF1 siRNA suppressed the expression of CRF1-LI in the NG. Subdiaphragmatic vagotomy decreased the number of CRF1-positive cells in the NG while it did not affect CRF-induced fecal output. CRF-induced c-Fos expression in the NTS was suppressed by vagotomy. A neuronal tracing study showed that approximately half of CRF1-positive cells expressed fast blue in the NG. Intraperitoneal CRF, a selective CRF1 agonist, or immobilization stress induced pCREB expression and increases in CRF1-positive cells in the NG. In contrast, a CRF1 antagonist reduced the immobilization-induced increase in the expression of pCREB in the NG. These results suggest that the CRF/CRF1 system is involved in the signal transduction of colonic sensory information to the central nervous system via the NG.

Diabetic heart disease remains the leading cause of death in individuals with type-2 diabetes mellitus (T2DM). Both insulin resistance and metabolic derangement, hallmark features of T2DM, develop early and progressively impair cardiovascular function. These factors result in altered cardiac metabolism and energetics, as well as coronary vascular dysfunction, among other consequences. Therefore, gaining a deeper understanding of the mechanisms underlying the pathophysiology of diabetic heart disease is crucial for developing novel therapies for T2DM-associated cardiovascular disease. Cardiomyocytes are equipped with the cholinergic machinery, known as the non-neuronal cardiac cholinergic system (NNCCS), for synthesizing and secreting acetylcholine (ACh) as well as possessing muscarinic ACh receptor for ACh binding and initiating signaling cascade. ACh from cardiomyocytes regulates glucose metabolism and energetics, endothelial function, and among others, in an auto/paracrine manner. Presently, there is only one preclinical animal model – diabetic db/db mice with cardiac-specific overexpression of choline transferase (Chat) gene - to study the effect of activated NNCCS in the diabetic heart. In this mini-review, we discuss the physiological role of NNCCS, the connection between NNCCS activation and cardiovascular function in T2DM and summarize the current knowledge of S-Nitroso-NPivaloyl-D-Penicillamine (SNPiP), a novel inducer of NNCCS, as a potential therapeutic strategy to modulate NNCCS activity for diabetic heart disease.

We previously reported a novel compound called S-nitroso-N-pivaloyl-d-penicillamine (SNPiP), which was screened from a group of nitric oxide donor compounds with a basic chemical structure of S-nitroso-N-acetylpenicillamine, to activate the nonneuronal acetylcholine system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The nonneuronal acetylcholine-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 hours after SNPiP administration) revealed that SNPiP initially induced Wnt and cyclic guanosine monophosphate-protein kinase G signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in response to SNPiP treatment, resulting in a metabolic shift from oxidative phosphorylation to glycolysis, which was suggested by reduced cardiac glucose contents while maintaining adenosine tri-phosphate levels. In addition, SNPiP significantly upregulated atrial natriuretic peptide and sarcolipin, which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

The reflexive excitation of the sympathetic nervous system in response to psychological stress leads to elevated blood pressure, a condition that persists even after the stress has been alleviated. This sustained increase in blood pressure, which may contribute to the pathophysiology of hypertension, could be linked to neural plasticity in sympathetic nervous activity. Given the critical role of astrocytes in various forms of neural plasticity, we investigated their involvement in maintaining elevated blood pressure during the post-stress phase. Specifically, we examined the effects of arundic acid, an astrocytic inhibitor, on blood pressure and heart rate responses to air-jet stress. First, we confirmed that the inhibitory effect of arundic acid is specific to astrocytes. Using c-Fos immunohistology, we then observed that psychological stress activates neurons in cardiovascular brain regions, and that this stress-induced neuronal activation was suppressed by arundic acid pre-treatment in rats. By evaluating astrocytic process thickness, we also confirmed that astrocytes in the cardiovascular brain regions were activated by stress, and this activation was blocked by arundic acid pre-treatment. Next, we conducted blood pressure measurements on unanesthetized, unrestrained rats. Air-jet stress elevated blood pressure, which remained high for a significant period during the post-stress phase. However, pre-treatment with arundic acid, which inhibited astrocytic activation, suppressed stress-induced blood pressure elevation both during and after stress. In contrast, arundic acid had no significant impact on heart rate. These findings suggest that both neurons and astrocytes play integral roles in stress-induced blood pressure elevation and its persistence after stress, offering new insights into the pathophysiological mechanisms underlying hypertension.

[This retracts the article DOI: 10.1016/j.heliyon.2023.e17434.].

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator‐activated receptor‐γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5 ′ untranslated region (5 ′UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (‐733 to ‐76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation‐quantitative PCR (ChIP‐qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR‐Cas9 system identified crucial PPARγ‐binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3‐L1 cells.

The non-neuronal cholinergic system of the cardiovascular system has recently gained attention because of its origin. The final product of this system is acetylcholine (ACh) not derived from the parasympathetic nervous system but from cardiomyocytes, endothelial cells, and immune cells. Accordingly, it is defined as an ACh synthesis system by non-neuronal cells. This system plays a dispensable role in the heart and cardiomyocytes, which is confirmed by pharmacological and genetic studies using murine models, such as models with the deletion of vesicular ACh transporter gene and modulation of the choline acetyltransferase (ChAT) gene. In these models, this system sustained the physiological function of the heart, prevented the development of cardiac hypertrophy, and negatively regulated the cardiac metabolism and reactive oxygen species production, resulting in sustained cardiac homeostasis. Further, it regulated extra-cardiac organs, as revealed by heart-specific ChAT transgenic (hChAT tg) mice. They showed enhanced functions of the blood-brain barrier (BBB), indicating that the augmented system influences the BBB through the vagus nerve. Therefore, the non-neuronal cardiac cholinergic system indirectly influences brain function. This mini-review summarizes the critical cardiac phenotypes of hChAT tg mice and focuses on the effect of the system on BBB functions. We discuss the possibility that a cholinergic signal or vagus nerve influences the expression of BBB component proteins to consolidate the barrier, leading to the downregulation of inflammatory responses in the brain, and the modulation of cardiac dysfunction-related effects on the brain. This also discusses the possible interventions using the non-neuronal cardiac cholinergic system.

Vascular endothelial cells play an important role in regulating peripheral circulation by modulating arterial tone in the microvasculature. Elevated intracellular Ca2+ levels are required in endothelial cells to induce smooth muscle relaxation via endothelium-dependent mechanisms such as nitric oxide production, prostacyclin, and endothelial cell hyperpolarization. It is well established that exogenous administration of acetylcholine can increase intracellular Ca2+ concentrations, followed by endothelium-dependent vasodilation. Although endogenous acetylcholine's regulation of vascular tone remains debatable, recent studies have reported that endogenously derived acetylcholine, but not neuronal cell-derived acetylcholine, is a key modulator of endothelial cell function. In this minireview, we summarize the current knowledge of the non-neuronal cholinergic system (NNCS) in vascular function, particularly vascular endothelial cell function, which contributes to blood pressure regulation. We also discuss the possible pathophysiological impact of endothelial NNCS, which may induce the development of vascular diseases due to endothelial dysfunction, and the potential of endothelial NNCS as a novel therapeutic target for endothelial dysfunction in the early stages of metabolic syndrome, diabetes, and hypertension.

Adult heart mostly contains long-lived postmitotic cardiomyocytes and non-cardiomyocytes that have proliferative potential. Here, we isolated cardiomyocytes and non-cardiomyocytes from young and aged mouse heart, and performed transcriptome analyses by RNA sequencing to understand the differences of gene expression in postmitotic and proliferative cells. Gene ontology analyses revealed that genes associated with inflammatory response were upregulated in aged cardiac myocytes, whereas genes including two ATP synthases in mitochondrial respiratory complex V ( Atp5l and Atp5J2 ) and two NADH dehydrogenases in complex I ( Ndufa11 and Ndufv3 ) were significantly downregulated. In aged non-cardiomyocytes, genes related to inflammatory responses were also upregulated, while genes involved in cell cycle and DNA replication process were downregulated. We also found that the expression levels of some small nucleolar RNAs (snoRNAs) are decreased cardiomyocytes with aging. snoRNAs are deeply involved in RNA modification such as pseudouridylation stabilizing ribosomal RNA (rRNA) and mRNA splicing. Therefore, the age-related reduction in snoRNA expression may lead to the destabilization of rRNA, splicing dysfunction, and ultimately a decrease in protein synthesis capacity. A comparison with transcriptome results obtained for non-cardiomyocytes suggests that the decline in the expression of mitochondria-related genes and snoRNAs accompanying aging is specific to cardiomyocytes, implying their potential utility as one of novel aging markers in postmitotic cells.

Low birthweight rats due to fetal undernutrition sustain higher corticosterone levels when exposed to stress. This is due to the upregulated expression of the pituitary-specific Gas5, a long noncoding RNA (lncRNA) that acts as a glucocorticoid receptor decoy and then competitively inhibiting the binding of glucocorticoids to DNA. However, the mechanism of Gas5 lncRNA upregulation remains unclear. Therefore, using the fetal undernourished model, we identified the factors that regulated Gas5 lncRNA expression and examined their effect on subsequent generations. We found that the expression levels of miR-23 was significantly lower in low birth-weight rats compared with controls. The expression of miR-23 was significantly lower and the expression levels of Gas5 lncRNA were significantly higher in the pituitary gland of low birth-weight offspring of the F2 and F3 generations compared with controls. The methyl modulator intervention in lactating F0 maternal rats restored miR-23 and Gas5 lncRNA expressions not only in F1, F2 and F3 offspring. Moreover, the intervention reduced circulating corticosterone levels and gene expressions in the pituitary gland after restraint stress exposure. In conclusion, miR-23-mediated alterations of the stress response are inherited and restored by methyl modulator intervention during lactation.

We have previously identified the novel splicing variant of mouse Pparγ ( Pparγ1sv ) and proposed the synergistic regulation of the early stage of adipocyte differentiation by Pparγ1sv and Pparγ2 . Here, we report the finding of PPARγ-binding sites within the Pparγ gene locus and its importance in adipogenesis and propose the positive feedback regulation of Pparγ1sv and Pparγ2 expression during the adipocyte differentiation of 3T3-L1 cells.

We previously developed a stress‐induced premature senescence (SIPS) model in which normal human fibroblast MRC‐5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar‐type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC‐5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short‐term co‐treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3‐chlorophenylhydrazone. By co‐treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt) were remarkably suppressed. Furthermore, AA co‐treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132‐treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.

Aims:Type 1 diabetes mellitus (T1DM) is associated with increased risk of cardiovascular disease (CVD) and mortality. The underlying mechanisms for T1DM-induced heart disease still remains unclear. In this study, we aimed to investigate the effects of cardiac non-neuronal cholinergic system (cNNCS) activation on T1DM-induced cardiac remodelling.Methods:T1DM was induced in C57Bl6 mice using low-dose streptozotocin. Western blot analysis was used to measure the expression of cNNCS components at different time points (4, 8, 12, and 16 weeks after T1DM induction). To assess the potential benefits of cNNCS activation, T1DM was induced in mice with cardiomyocyte-specific overexpression of choline acetyltransferase (ChAT), the enzyme required for acetylcholine (Ac) synthesis. We evaluated the effects of ChAT overexpression on cNNCS components, vascular and cardiac remodelling, and cardiac function.Key findings:Western blot analysis revealed dysregulation of cNNCS components in hearts of T1DM mice. Intracardiac ACh levels were also reduced in T1DM. Activation of ChAT significantly increased intracardiac ACh levels and prevented diabetes-induced dysregulation of cNNCS components. This was associated with preserved microvessel density, reduced apoptosis and fibrosis, and improved cardiac function.Significance:Our study suggests that cNNCS dysregulation may contribute to T1DM-induced cardiac remodelling, and that increasing ACh levels may be a potential therapeutic strategy to prevent or delay T1DM-induced heart disease.