Substance P/neurokinin1 receptor is associated with the expression of cardiac stem/pluripotency-associated genes in diabetic right and left atria

Substance P/neurokinin1 receptor is associated with the expression of cardiac stem/pluripotency-associated genes in diabetic right and left atria. Abstract Substance P (SP) is a cardioprotective neuropeptide that interacts with the G protein-coupled neurokinin-1 receptor (NK 1 R). Although the expression of SP/NK 1 R in the right atrium (RA) of diabetic patients is known to be significantly impaired, the molecular mechanism remains unclear. LETO and OLETF rats were randomly divided into 3 groups: saline, SP injection (5 nmole/kg), and SP+RP injection (1 mg/kg RP67580, a selective non-peptide tachykinin NK 1 R antagonist). After 3 weeks, the left atrium (LA), RA, and left ventricle (LV) of the rats were collected. Cardiac stem/pluripotency-associated genes in the diabetic atria of each group were comprehensively examined using qRT-PCR analysis. qRT-PCR analysis demonstrated that only the RA of SP-treated OLETF rats exhibited significantly higher levels of  -SMA, GATA4, TBX5, and Klf4 in the mRNA, compared to the control. RP prevented the expression of NK 1 R and four SP-associated genes in the RA of SP-treated OLETF rats. In conclusion, our findings provide novel mechanistic insights into the role of NK 1 R in diabetic atria. local activation of progenitor cells (CSCs) and RA 10,12 on how SP/NK 1 R relates biomarkers and molecular mechanisms between DM and atria. The present study was performed to identify the of genes between LETO and OLETF rats or presence or absence and to assess SP’s potential relevance as a novel biomarker and its role as a mechanism between atria and the diabetic heart.


Introduction
The relative risk of cardiovascular disease (CVD) is much higher for individuals with diabetes mellitus (DM) 1,2 . In recent years, there has been an increased interest in validating and discovering more about known biomarkers of CVD in such individuals [1][2][3] . Prognostic biomarkers and biomarker combinations could affect processes of care and improve patient outcomes 3 . In general, the majority of DM research focuses on the left ventricle (LV) 4,5 . DM can directly affect LV mass, hypertrophy, remodeling, and function 4,5 . Recently, several studies have recognized relationships between DM and the atria [6][7][8] . For example, one study compared the structure and function of the right atrium (RA) of individuals with normal glucose metabolism, prediabetes, and diabetes. Using the Maastricht Study, an observational prospective population-based cohort study, two-dimensional and tissue Doppler echocardiography, and multiple linear regression analyses, the researchers found (pre)diabetes to be associated with structural RA and right ventricle (RV) changes, as well as impaired RV systolic and diastolic function 6 .
Advanced anatomic and pathologic imaging techniques including echocardiography, computed tomography, chest radiography, and magnetic resonance imaging have provided a better depiction of the atria, which has improved objective assessment of clinical signs and symptoms of the RA 9,10 . Although these techniques are not ordinarily applied to identify the molecular mechanisms underlying diabetes-induced cardiac damage, a recent study used electron miscroscopy paired with morphemetric analysis to investigate the effects of diabetes on RA cardiomyocytes in Wistar elderly rats 11 . Compared to the control group, the rats with streptozotocin-induced diabetes showed increased functional activity of atrial cardiomyocytes with greater production of natriuretic peptides 11 . However, biomarkers and the underlying molecular mechanisms connecting DM and the atria remain elusive.
Substance P (SP), an 11-amino acid neuropeptide of the tachykinin neuropeptide family, 4 is an evolutionarily older neurotransmitter than acetylcholine and the catecholamines 10,12,13 .
Neurokinin1 receptor (NK1R), a G protein coupled receptor found in the central and peripheral nervous systems, is important for the biological actions of SP 10,12,13 . Interestingly, the expression and localization of SP and NK1R have already been described for RA of diabetic patients and rat models, shown to be significantly impaired 13,14 . Our recent, previous studies have demonstrated that treatment of SP enhanced the local activation of cardiac progenitor c-Kit + cells (CSCs) in LV and RA after myocardial infarction 10,12 . Based on these studies, we turn to unresolved key questions about how SP/NK1R relates to biomarkers and molecular mechanisms between DM and atria. The present study was performed to identify the expression of CSC-associated genes between LETO and OLETF rats with or without SP in the presence or absence of RP67580, and to assess SP's potential relevance as a novel biomarker and its role as a mechanism between atria and the diabetic heart.

OLETF RP+SP exhibited lower LVW/BW and RAW/BW ratios compared to both OLETF saline and OLETF SP , a reduction associated with the -SMA and NK1R expression
Several studies have highlighted altered patterns of abnormality affecting the size, shape, and function of hearts in patients with DM 6,7 . A previous paper reported that RA of diabetic patients exhibited decreased expression of NK1 14 . If SP/NK1R signaling contributes to cardiac abnormality, changes in the heart size of patients with DM could be explained by a link between AF and DM. To test this, we measured the ratio of LV, RA, and LA per BW in the LETO and OLETF groups. As shown in Figure 1B and 1C, compared to the OLETF saline group, LVW/BW was significantly lower in the OLETF SP (P<0.05) and OLETF RP+SP (P<0.01) groups, while the LVW/BW ratio of the LETO SP group was not significantly different from the LETO saline group. After SP injection RAW/BW and LAW/BW both increased for the LETO SP group, while only LAW/BW of OLETF SP experienced a marked increase ( Fig. 2B and 2C). Unexpectedly, we found that the mean RAW/BW ratio of the OLETF RP+SP group was significantly lower than for RA of the OLETF saline and OLETF SP groups. The weight of LA in the OLETF SP group slightly increased, whereas the weight decreased in the presence of RP ( Fig. 2D and 2E).
If SP/NK1R signaling correlates to change in the size of LA and RA, atrial remodeling could be caused by interstitial fibrosis, which is characterized by the myogenic properties of myofibroblasts. To test this, we further examined whether SP/NK1R signaling affects the expression of -SMA mRNA, which is used as a fibrosis marker 7 , 11 in the RA and LA of the LETO and OLETF groups. As shown in Figure 3A, up-regulation of -SMA mRNA was found only in the RA of OLETF SP . Inactivation of NK1R by RP inhibited the SP-mediated -SMA expression in RA of OLETF SP. On the other hand, the expression of -SMA had no effect on the RA of LETO saline and LETO SP , nor on the LA of OLETF saline , OLETF SP , and OLETF RP+SP 6 ( Fig. 3B). The expression pattern of NK1R mRNA was similar to -SMA in RA of OLETF SP (Fig. 3C). In the LA of the LETO and OLETF groups, NK1R mRNA showed oppositional and low expression compared with RA in the LETO and OLETF groups (Fig. 3D). These observations indicate that SP/NK1R signaling may correlate to atrial size and fibrosis in diabetic rats.

SP/NK1R signaling is involved in CSC-related gene expression in OLETF models
The number of c-Kit + cells derived from LA appendages in the atrial fibrillation (AF) heart is much lower than in a healthy heart 15 . In fact, c-Kit + cells are more abundant in the RA appendages 10 . Our previous studies have noted the local activation of c-Kit + cells by SP/NK1R signaling in RA in heart damage 10 . According to a recent study that constructed a rat model of right heart disease (RHD), RA can play an important role in AF maintenance with RA re-entrant activity caused by atrial fibrosis and conduction abnormalities 6 compared to the OLETF saline or OLETF RP+SP groups (Fig. 4, Fig. 5, and Fig. 6). The expression of NK1R-associated SCA1, c-KIT, NANOG, and OCT4 in the LA of the OLETF SP group was 7 of a similarly low level as the expressions of these SP-associated genes in the RA of the OLETF RP+SP group (Fig. 5 and Fig. 6). These findings suggest that SP/NK1R signaling correlates with CSC-related gene expression in RA of OLETF groups.

DISCUSSION
Our study is the first comprehensive study analyzing the biological role of SP/NK1R on CSC-related gene expression between the RA and LA of LETO and OLETF rats. The present study highlights changes in CSC and transcription factor related gene expression underlying RA and the diabetic heart. We found that SP and/or SP/RP administration reduced the weight of LV in the OLETF group, whereas there was no difference in LV weight between the LETO saline and LETO SP group. LV dysfunction in DM generally increases LA pressures, causing atrial remodeling 16,17 . Accordingly, compared to the LA in the LETO saline group, the LA weight of the LETO and OLETF groups slightly increased by SP. Unexpectedly, RA of the LETO SP group seemed to have increased, while the RA weight in the OLETF SP group remained the same as the control group and RP significantly reduced in the OLETF RP+SP group.
To explain these findings, we focused on the gene expression of endogenous c-Kit + CSCrelated markers and a subset of activated fibrogenic cells and myofibroblasts in atrial remodeling, including fibrosis 19 . This focus was informed by our previous studies demonstrating the significance of SP/NK1R signaling in local activation of c-Kit + CPCs of RA and LV following heart damage 10,12 . Furthermore, a recent paper reported the impact of streptozotocin-induced diabetes on gene expression and DNA methylation in cardiac cells (20). Of note, cell type-specific analysis shows differentially modulate gene programs that are involved in distinct biological processes in diabetes 20 . GATA4, a zinc finger transcription factor, can activate the promoters of many cardiacspecific genes such as SMADs, NKX2-5, and TBX5 26  and AccuPower®ProFi Taq PCR PreMix were purchased from Bioneer (DaeJeon, Korea).
SYBR®Green Mix was obtained from Applied Biosystems (Lincoln, CA, USA).

Experimental animals
All procedures for animal models in this study were approved by the Institutional Animal Care Center standardized pain protocol, all LETO and OLETF rats continually was monitored for signs of distress. The LETO and OLETF rats were housed in the same pathogen-free facility under a 12 h light and dark cycle with ad libitum feeding. No more than two animals were housed per cage 10,12 .
Experimental design, sample collection, and measurement of LA, RA, LV, and body weight ratio As shown in Fig. 1A, the rats were randomly divided into 5 groups (n=4 each); LETO (LETO saline ), LETO with 5 nmole/kg SP injection (LETO SP ), OLETF saline , OLETF with 5 nmole/kg SP injection (OLETF SP ), and OLETF SP with a 1 mg/kg injection of RP67580 (OLETF RP+SP ). SP and RP were injected twice into the tail vein of the OLETF SP at 27 and 28 weeks. At 29 weeks, the rats were anaesthetized by using isoflurane (Hana Pharm Co.,Ltd., Seoul, Korea), and then were euthanized. The hearts were gently grasped with blunt forceps, then punctured with a 21 G needle to aspirate blood from LV. The hearts were washed with PBS and then LA, RA, and LV tissue samples were collected ( Fig. 2A). LA mass, RA mass, LV mass, and body weight were measured using an electronic balance (model GR-200 and CUX220H). The percentage of each weight ratio was calculated as follows: the ratio of LA, RA, or LV = (LA, RA, or LV weight/body weight) x %. The weight for every sample was immediately measured and then frozen in liquid nitrogen and stored at -80°C.

Statistical analysis
A one-way analysis of variance (ANOVA) (for comparisons between three or more groups) or student's t-tests (for comparisons of two groups) or followed by Tukey post hoc tests were used for the statistical analyses. GraphPad Prism software (GraphPad Software Inc) and SPSS software ver. 17.0 (SPSS, Chicago, IL) were used to determine the statistical analysis. A value of P < 0.05 was considered significant. Data are expressed as means ± standard error (SD). *P < 0.05 -0.01, **P < 0.01 -0.001, and ***P < 0.001 vs. correspond ing LETO saline and/or OLETF saline . All error bars mean the standard deviation of three or more biological replicates of indicated data.