Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-nitroso-N-pivaloyl-D-penicillamine (SNPiP): Implications for Improved Diastolic Function and Cardiac Performance

We previously reported a novel compound called S-nitroso-N-pivaloyl-D-penicillamine (SNPiP), which was screened from a group of nitric oxide (NO) donor compounds with a basic chemical structure of S-nitroso-N-acetylpenicillamine (SNAP), to activate the non-neuronal acetylcholine (NNA) system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The NNA-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 h after SNPiP administration) revealed that SNPiP initially induced Wnt and cGMP-protein kinase G (PKG) 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 SNPiP-treated samples, indicating a metabolic shift from oxidative phosphorylation to glycolysis following SNPiP administration in the hearts. Additionally, SNPiP significantly upregulated atrial natriuretic peptide (ANP) and sarcolipin (SLN), 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 non-neuronal acetylcholine (NNA) system, which is distinct from the parasympathetic 3 nervous system (PNS), is a unique effector that influences local physiological functions in an 4 autocrine/paracrine manner. The non-neuronal cardiac cholinergic system (NNCCS), which is a 5 component of the NNA system, is present within cardiomyocytes and is responsible for the 6 synthesis of acetylcholine (ACh) independently of the PNS [1][2][3]. Therefore, in the heart, ACh is 7 derived from two sources: the PNS and the NNCCS. Using murine models with gain and loss of 8 function, we and other groups have already reported that the NNCCS is a crucial system to 9 sustain cardiac functions and provide resilience potential against hypoxia/ischemia by 10 upregulating survival signals, modulating energy metabolism preference, enhancing 11 angiogenesis, and activating cell-cell communication [4][5][6][7]. These findings prompted us to 12 explore the development of an inducer or activator of the NNCCS. Based on the recognition that 13 the NO donor SNAP exhibits relatively weak potency in inducing choline acetyltransferase 14 (ChAT), we screened chemical compounds with a basic structure similar to that of SNAP.

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NO is naturally produced in the body and has the ability to dilate blood vessels. NO also has 16 many physiological functions, including regulation of blood pressure and protection of the heart, 17 which has led to the development of NO-based therapies for heart disease [8,9]. However, 18 acute dilation of blood vessels can decrease blood pressure and concomitantly increase heart 19 rate in a compensatory manner. Moreover, despite the effectiveness of NO-based drugs in the 20 treatment of heart disease, their short half-lives make their use difficult. The half-lives of 21 N-diazeniumdiolates (NONOates) is from 2 s to 20 h [10], whereas that of SNAP is 1 approximately 6 h [11]. Therefore, the development of a novel NO-generating drug that causes 2 sufficient and long-lasting NO release with less of an effect on hemodynamic parameters and 3 that ultimately activates the NNCCS can provide a novel therapeutic modality that differs from 4 conventional modalities.

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We previously reported the effects of SNPiP, a novel NO donor and NNCCS activator, on cardiac 6 function [12]. This compound releases NO for longer than traditional NO donors, indicating its 7 potential to enhance cardiac function. Indeed, in vivo experiments with SNPiP-treated mice have 8 demonstrated improved diastolic function without tachycardia, leading to enhanced cardiac 9 output without an increase in heart rate which supports our hypothesis that the NNCCS plays a 10 crucial role in sustaining cardiac functions, including potentiating resilience to ischemia, 11 modulating energy metabolism preference, and activating angiogenesis [12]. SNPiP also 12 improved impaired cardiac function in db/db hearts [13], suggesting its potential use as a 13 therapeutic agent to activate the NNCCS and modulate cardiac function. It has been reported that 14 SNPiP gradually elevates the intracellular levels of cyclic guanosine monophosphate (cGMP) and 15 NO levels in H9c2 and HEK293 cells [12], which in turn upregulate ChAT and increase ACh 16 synthesis. However, the detailed molecular mechanisms by which SNPiP affects cardiac function 17 remain unclear.

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To elucidate how SNPiP influences the expression of genes associated with cardiac function, we 19 conducted a transcriptome analysis of ventricles from wild-type male mice treated with SNPiP.

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Gene Ontology (GO) analysis revealed that SNPiP upregulated genes involved in cardiac 21 muscle development and cellular differentiation. Furthermore, the results of pathway analysis 6 1 suggested that SNPiP activates the oxytocin signaling pathway, which is known to improve 2 cardiac performance and stimulate cardiomyocyte differentiation. Lastly, we found that 3 administration of SNPiP resulted in significant upregulation of atrial natriuretic peptide (ANP) 4 and sarcolipin (SLN), which could be crucial for elucidating the comprehensive mechanisms of 5 the effect of SNPiP on cardiac function. Our study provides a more in-depth understanding of 6 the molecular mechanisms underlying the therapeutic effects of NO-based drugs and their 7 potential adverse effects. SNPiP, which has a molecular weight of 262.32, was synthesized using a previously reported 12 method [12]. This compound, identified by product code: 197-19151, is now commercially 13 available and can be procured from FUJIFILM Wako Chemicals (Osaka, Japan). Adult male 14 C57/BL6 mice aged 12 weeks were used in the experiments. SNPiP was dissolved in dimethyl 15 sulfoxide (DMSO) (D2650, Sigma-Aldrich Japan, Tokyo, Japan) and administered by 16 intraperitoneal injection at a dose of 60 pmol/g body weight. As a control, a comparable dilution 17 of the solvent was administered in the same manner [12]. After an appropriate amount of time, 18 the heart was removed from a mouse euthanized by cervical dislocation. The atria were 19 subsequently dissected from the heart, leaving the ventricles, which were then used as samples.

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Total RNA was isolated from the whole ventricle of mice using ISOGEN reagent (Nippon

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Gene, Tokyo, Japan) according to the manufacturer's instructions. The yield and quality of the 7 1 purified RNA were evaluated by spectrophotometry, agarose gel electrophoresis, and 2 Bioanalyzer assessments. The library preparation and RNA-seq were conducted by Macrogen 3 Japan (Tokyo, Japan) using a TruSeq TM Stranded Total RNA Library Prep Kit with a 4 Ribo-Zero TM kit and NovaSeq6000 paired-end sequencing (Illumina). The sequencing reads 5 were aggregated into an rRNA reference (Mouse_rRNA_Reference_BK000964) using Bowtie 6 package to remove rRNA reads. Unpaired reads were removed using FASTQ-pair. Paired reads 7 were aligned using STAR version 2.7.8a with the GRCm38 reference alignment. RSEM was 8 used to quantify the transcript. Transcripts Per Million (TPM) calculated using RSEM were 9 log 2 (count+1) transformed.

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Significance estimates of differences in gene expression, such as the p-value and False

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Discovery Rate (FDR), were calculated from expected counts using edgeR package in R.

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Differentially expressed genes (DEGs) were defined as genes with a p-value < 0.05 and |log 2 (fold   Using TMM values, which are normalized count data, GSEA was performed according to the 5 methods described on the GSEA website (http://www.gsea-msigdb.org/gsea/index.jsp).

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Western blot analysis 7 Tissue lysates were prepared from cardiac ventricles excised from the heart using T-PER TM 8 Tissue Protein Extraction Reagent (ThermoFisher Scientific, Tokyo, Japan) according to the 9 manufacturer's instructions. After centrifugation, the tissue lysate supernatant was collected and 10 treated with SDS sample buffer. Comparable amounts of protein (10-30 g/lane), quantified 11 using a BCA kit, were loaded (as subsequently verified based on the level of GAPDH). Tissue 12 lysate supernatants were electrophoresed and blotted onto polyvinylidene fluoride membranes.

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After blocking with 4% skim milk, the membranes were incubated with the following primary

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Transcriptome analysis of SNPiP-administered ventricles of wild-type mice 9 1 Male wild-type C57/BL6 mice were intraperitoneally administered with SNPiP dissolved in 2 DMSO (Fig. 1A). Male mice were used to exclude sexual dimorphism particularly the 3 beneficial effects of estrogen on cardiovascular diseases. The time points for sacrificing 4 SNPiP-treated mice (24 and 48 h) were determined based on our previous studies. In these 5 studies, the effect of SNPiP on cardiac dilation was most pronounced 48 and 72 h after 6 administration [12,13]. To investigate how SNPiP affects the expression of genes related to 7 cardiac function, changes in gene expression in wild-type mice treated with DMSO (control) or 8 SNPiP were determined by RNA-seq.

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We conducted PCA to visualize the differences and similarities between the samples (Fig. 1C).

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Plotting of the data onto the first two principal components revealed that the SNPiP-treated

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Pathway analyses 20 KEGG pathway analysis revealed that enriched pathways mainly involved "Wnt signaling 21 pathway" and "cGMP-PKG signaling pathway" were remarkably affected by the administration 22 of SNPiP at 24 h (Fig. 3A), compared with the DMSO controls. Figure 3B illustrates KEGG 1 pathway mapping for "Wnt signaling pathway". More than 10 genes in the canonical Wnt 2 pathway were upregulated, while six genes in the Wnt/Ca 2+ pathway were also identified ( Fig.   3 3B), implying that SNPiP triggered Ca 2+ signaling, which may be involved in regulating cardiac 4 contraction and dilation. The analysis also identified that "oxytocin signaling" was enriched at 5 both 24 and 48 h (Fig. 3A). Ingenuity pathway analysis (IPA) also revealed significant 6 activation of multiple signaling pathways, including the "Wnt/Ca 2+ pathway" and "oxytocin 7 signaling pathway" in addition to other molecular signaling pathways such as "estrogen receptor 8 signaling", particularly at 24 h after SNPiP administration (Fig. 3C). Differential gene 9 expression in the "oxytocin signaling pathway" was visualized by hierarchical clustering 10 heatmap analysis (Fig. 3D), and the IPA pathway map (Fig. 3E). Although the induction levels  We identified highly upregulated (log 2 FC > 1) and downregulated (log 2 FC < -1) genes by 2 SNPiP at 24 and 48 h (p < 0.05), some of which were common to both time points (Figs. 4A 3 and 4B). These shared genes play important roles in the response to hypoxia, tissue 4 development, Ca 2+ homeostasis, and the regulation of muscle relaxation (Table 1). Among 5 them, Nppa encodes the cardiac hormone ANP, which promotes vasodilation, reduces heart 6 workload and lowers blood pressure. We confirmed the significant upregulation of ANP levels in 7 the SNPiP-treated murine ventricles by immunoblotting (Fig. 4C). Similarly, Sln encodes SLN, 8 which plays a significant role in cardiac function, specifically in the regulation of Ca 2+ handling 9 by interaction with the sarcoplasmic/endoplasmic reticulum ATPase (SERCA) pump in 10 cardiomyocytes. Upregulation of SLN levels by SNPiP was also evaluated by immunoblotting 11 (Fig. 4D). Lastly, we also identified the protein-protein interaction networks associated with 12 highly differentially expressed transcripts using STRING analysis. While many interactions were 13 attributed to the co-expression of genes in publicly available gene expression datasets, physical

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In this report, we show the time-course effect of a novel NO donor, SNPiP, on the cardiac gene 20 expression profile of wild-type mouse ventricles. In SNPiP-treated mice, diastolic function was 21 greatly enhanced, suggesting increased left ventricular compliance, resulting in improved 22 cardiac output, probably through the Frank-Starling law; hence, the drug potentially provides a 13 1 new treatment strategy for cardiovascular diseases. Although the specific effects of SNPiP on 2 protein levels, modification, and interaction remain to be elucidated, our transcriptome analysis 3 revealed that SNPiP treatment promotes heart tissue development and cardiomyocyte 4 differentiation. The terms annotated in the enrichment analysis primarily relate to embryonic 5 heart development. However, the adult heart is capable of repair and regeneration [14].

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Therefore, they can also be applied to certain aspects of heart repair and regeneration, along

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Another NO donor, SNAP, also promotes cardiac differentiation in mouse embryonic stem (ES) 12 cells [16]. Thus, we hypothesized that the NO produced by SNPiP triggers cardiogenesis and 13 beneficial remodeling in the heart, resulting in improved cardiac function (Fig. 4F). Moreover,

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we previously reported that SNPiP induces NNCCS, leading to a gradual increase in ACh 15 synthesis in cardiomyocytes [8]. This in turn upregulates NO production in an 16 autocrine/paracrine manner, and NO from the donor alternatively upregulates the 17 hypoxia-responsive pathway, as described later [17,18]. Taken together, the enhanced NO      In addition to Nppa and Sln, we identified several genes that are highly upregulated following 2 SNPiP administration (Figs. 4A and 4B). Intriguingly, four of these genes (Nppa, Sln, Myl4, and shown), we believe that the similarities in the gene expression profiles between our model and 7 the ET B receptor study imply a common mechanism influencing cardiac function. Indeed, GO 8 analysis revealed enrichment in the BP term "response to hypoxia" in SNPiP-treated ventricles at 9 48 h (p = 3.83 × 10 -6 ) (Fig. 1E). The significant differential regulation of four key genes involved 10 in hypoxia signaling Egln3, Vegfa, Nppa, and Angptl4 (Fig. 1H), which are among the top 25 11 genes with high hypoxia-normoxia scores [33], supports the hypothesis that SNPiP induces  Of note, the top-scored network among counterregulated genes, including Nppa, Sln, and Myl4, 2 in EdnrB +/+ and EdnrB -/+ mice is related to the cGMP metabolic process and its connection to 3 Ca 2+ homeostasis [32]. NO upregulates cGMP, the second messenger of NO, in the heart, which 4 activates PKG and eventually regulates Ca 2+ homeostasis by either enhancing Ca 2+ release via 5 ryanodine receptors from the sarcoplasmic reticulum or by inhibiting L-type calcium channel 6 [38]. We found that SNPiP increased cGMP levels in HEK293 cells [12] and activated the 7 cGMP-PKG signaling pathway in vivo (Fig. 3A). Therefore, the regulation of Ca 2+ homeostasis 8 through cGMP-PKG signaling appears to be another mechanism shared between our model and 9 the ET B receptor study. However, the ET B receptor plays a crucial role in mediating NO

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Our previous studies demonstrated that SNPiP possesses the characteristic advantage of 21 inotropic action by enhancing cardiac dilatation without inducing tachycardia [12]. This is in 22 contrast to conventional inotropic agents, which enhance oxygen consumption and require more 1 energy substrate resulting in a substantial cardiac workload. Furthermore, the mode of action of 2 SNPiP is expected to be indolent based on the production mode of cGMP and NO consequently 3 enhancing ACh production through NNA and NNCCS. This is supported by the observation 4 that SNPiP enhances murine cardiac function to its maximum extent within 48 to 72 h [12]. As 5 shown in this study, SNPiP triggers the expression of various genes involved in multilateral 6 functions, and de novo protein synthesis induced by SNPiP may be integrated to perform such a 7 cardioprotective action. In contrast, it is important to note that SNPiP is administered 8 intravenously rather than orally. Consequently, exploring the potential of modifying SNPiP for 9 oral delivery could offer significant advantages for future clinical applications.