Abstract

   Diabetic cardiomyopathy (DCM), one of the most serious long-term
   consequences of diabetes, is closely associated with myocardial fatty
   acid metabolism. Carnitine palmitoyltransferase-1β (CPT-1β) is the
   rate-limiting enzyme responsible for β-oxidation of long-chain fatty
   acids. Intermedin (IMD) is a pivotal bioactive small molecule peptide,
   participating in the protection of various cardiovascular diseases.
   However, the role and underlying mechanisms of IMD in DCM are still
   unclear. In this study, we investigated whether IMD alleviates DCM via
   regulating CPT-1β. A rat DCM model was established by having rats to
   drink fructose water for 12 weeks. A mouse DCM model was induced by
   feeding mice a high-fat diet for 16 weeks. We showed that IMD and its
   receptor complexes levels were significantly down-regulated in the
   cardiac tissues of DCM rats and mice. Reduced expression of IMD was
   also observed in neonatal rat cardiomyocytes treated with palmitic acid
   (PA, 300 μM) in vitro. Exogenous and endogenous IMD mitigated cardiac
   hypertrophy, fibrosis, dysfunction, and lipid accumulation in DCM rats
   and IMD-transgenic DCM mice, whereas knockout of IMD worsened these
   pathological processes in IMD-knockout DCM mice. In vitro, IMD
   alleviated PA-induced cardiomyocyte hypertrophy and cardiac fibroblast
   activation. We found that CPT-1β enzyme activity, mRNA and protein
   levels, and acetyl-CoA content were increased in T2DM patients, rats
   and mice. IMD up-regulated the CPT-1β levels and acetyl-CoA content in
   T2DM rats and mice. Knockdown of CPT-1β blocked the effects of IMD on
   increasing acetyl-CoA content and on inhibiting cardiomyocyte
   hypertrophy and cardiac fibroblast activation. IMD receptor antagonist
   IMD[17–47] and the phosphatidyl inositol 3 kinase (PI3K)/protein kinase
   B (Akt) inhibitor LY294002 reversed the effects of IMD on up-regulating
   CPT-1β and acetyl-CoA expression and on inhibiting cardiomyocyte
   hypertrophy and cardiac fibroblast activation. We revealed that IMD
   alleviates DCM by up-regulating CPT-1β via calcitonin receptor-like
   receptor/receptor activity-modifying protein (CRLR/RAMP) receptor
   complexes and PI3K/Akt signaling. IMD may serve as a potent therapeutic
   target for the treatment of DCM.

   Keywords: diabetic cardiomyopathy, intermedin, CPT-1β, PI3K/Akt

1. Introduction

   Diabetes has become one of the most prevalent and serious chronic
   diseases, and it is a metabolic disorder characterized by
   hyperglycemia, resulting from impaired insulin secretion and/or action
   [[46]1,[47]2]. One of the most common complications of diabetes
   mellitus is diabetic cardiomyopathy (DCM), which is the leading cause
   of death in diabetic patients [[48]3,[49]4,[50]5,[51]6]. DCM refers to
   cardiac structural and functional abnormalities caused by disturbance
   in myocardial glucose and lipid metabolism in diabetic patients,
   regardless of other factors such as coronary artery disease, valvular
   disease, and hypertension [[52]2,[53]4]. DCM is characterized by
   cardiac hypertrophy, cardiac fibrosis, several perturbations in
   myocardial energy metabolism, and left ventricular (LV) diastolic
   dysfunction with normal ejection fraction (EF) evolving to LV systolic
   dysfunction with reduced EF [[54]6,[55]7,[56]8,[57]9]. Increased fatty
   acid uptake surpasses fatty acid oxidation, resulting in a large amount
   of lipid accumulation and overload in the diabetic heart. These
   pathophysiological abnormalities cause LV diastolic and systolic
   dysfunction, promoting the occurrence and development of DCM
   [[58]10,[59]11,[60]12]. Increasing myocardial fatty acid oxidation may
   offer a potential strategy to prevent DCM or heart failure via reducing
   lipid accumulation [[61]11,[62]12,[63]13]. Currently, there are no
   effective therapeutic targets to protect against DCM. Therefore, it is
   urgent to discover promising therapeutic targets for DCM.

   Carnitine palmitoyltransferase-1 (CPT-1) is the rate-limiting enzyme
   responsible for β-oxidation of long-chain fatty acids. CPT-1 has two
   isoforms, CPT-1α and CPT-1β, that are located in the outer
   mitochondrial membrane. CPT-1β is the primary CPT-1 isoform in the
   heart. CPT-1β mediates the transport of fatty acyl CoA into the
   mitochondria for myocardial fatty acid β-oxidation and then generates
   acetyl-CoA [[64]14,[65]15,[66]16,[67]17,[68]18]. A previous report
   demonstrated that CPT-1β expression was elevated in the heart of T2DM
   mice [[69]17]. Overexpression of CPT-1 can enhance fatty acid
   oxidation, reduce lipid accumulation, and improve insulin sensitivity
   in high-fat-diet (HFD)-fed rats [[70]18]. Deletion or inhibition of
   CPT-1β can lead to myocardial lipid accumulation, reduced insulin
   sensitivity, cardiac hypertrophy, cardiac fibrosis, and cardiac
   dysfunction in mice [[71]19,[72]20,[73]21,[74]22,[75]23]. These
   findings suggest that up-regulating CPT-1β might be a possible
   therapeutic target for alleviating DCM.

   Many studies have demonstrated that multiple bioactive molecules can
   regulate CPT-1β expression, such as leptin [[76]24], adiponectin
   [[77]25], and eicosapentaenoic acid [[78]26] up-regulating the mRNA
   levels of Cpt1b in the hearts of rats, C[2]C[12] myocytes, and primary
   human adipose-tissue-derived stem cells, respectively. By contrast,
   angiotensin II [[79]27] down-regulates the protein levels of CPT-1 in
   adult rat cardiomyocytes. Intermedin (IMD), also known as
   adrenomedullin 2 (ADM2), is a member of the calcitonin gene-related
   peptide (CGRP) superfamily discovered in 2004, which is a
   paracrine/autocrine bio-active polypeptide [[80]28,[81]29]. IMD[1–53]
   is the longest active fragment, which is produced by proteolytic
   cleavage of human prepro-IMD [[82]30,[83]31,[84]32,[85]33,[86]34]. IMD
   exists in various cardiovascular cells, including cardiomyocytes,
   fibroblasts, vascular smooth muscle cells, and endothelial cells
   [[87]30,[88]31,[89]32,[90]33,[91]34]. IMD binds non-selectively to its
   receptor complexes, calcitonin receptor-like receptor/receptor
   activity-modifying proteins (CRLR/RAMPs), and activates post-receptor
   signaling pathways to exert biological effects
   [[92]30,[93]31,[94]32,[95]33,[96]34]. IMD has a protective role in
   various cardiovascular diseases, such as cardiac hypertrophy, cardiac
   fibrosis, congestive heart failure, hypertension, atherosclerosis, and
   vascular calcification [[97]30,[98]34,[99]35,[100]36,[101]37,[102]38].
   Moreover, IMD can reduce the levels of fasting blood glucose (FBG),
   insulin, free fatty acid, triglycerides, and total cholesterol, thereby
   improving metabolic syndromes like obesity and insulin resistance in
   T1DM rats, T2DM mice, hyperhomocysteinemia mice, and ApoE^−/−
   atherosclerosis mice [[103]39,[104]40,[105]41,[106]42]. These results
   indicate that IMD may serve as a potent therapeutic target for the
   treatment of DCM. However, the role and possible mechanism of IMD in
   DCM remain unclear.

   In this study, we investigated the role and mechanism of IMD in DCM. We
   found that IMD expression is down-regulated in heart tissues of DCM
   rats and mice. Exogenous and endogenous IMD mitigated DCM, whereas
   knockout of IMD worsened DCM. Mechanistically, this study identified
   that IMD alleviates DCM by up-regulating CPT-1β via CRLR/RAMP receptor
   complexes and phosphatidyl inositol 3 kinase (PI3K)/protein kinase B
   (Akt) signaling, suggesting that targeting IMD is a potential
   therapeutic strategy for DCM.

2. Results

2.1. IMD Levels Are Significantly Down-Regulated in Cardiac Tissues of DCM
Rats, DCM Mice, and Hypertrophic Cardiomyocytes

   We established a rat DCM model by having rats to drink fructose
   drinking water for 12 weeks. To reveal the potential role of exogenous
   IMD in DCM, we first examined the expression of IMD and its receptor
   complexes in the cardiac tissues of fructose-drinking rats. Cardiac
   mRNA levels of IMD and mRNA and the protein levels of its receptor
   complexes, CRLR/RAMPs, were decreased in fructose-drinking rats
   ([107]Figure S1A,B). In vitro, we observed palmitic acid (PA)-induced
   cardiomyocyte hypertrophy ([108]Figure S1C) and cardiac fibroblast
   activation of rats ([109]Figure S1D). The IMD mRNA levels were
   decreased in PA-treated neonatal rat cardiomyocytes (NRCMs)
   ([110]Figure S1E). These findings indicated that IMD is involved in DCM
   in fructose-drinking rats through its receptor complexes. We also
   examined the expression of IMD and its receptor complexes in the
   cardiac tissues of HFD-fed wildtype (WT) mice. Cardiac mRNA levels of
   IMD and mRNA and the protein levels of its receptor complexes,
   CRLR/RAMPs, were decreased in HFD-fed WT mice ([111]Figure S2A,B),
   indicating that IMD is involved in DCM in HFD-fed mice through its
   receptor complexes.

2.2. Exogenous IMD Alleviates DCM in Rats

   Firstly, we constructed the DCM model by using fructose-drinking rats
   and assessed the influence of exogenous IMD on rat DCM in different
   dimensions. IMD treatment significantly reduced
   fructose-drinking-induced cardiac hypertrophy in rats, as shown by a
   reduced myocardial cross-sectional area and down-regulated mRNA levels
   of pro-hypertrophic genes natriuretic peptide A (Nppa) and natriuretic
   peptide B (Nppb) ([112]Figure 1A,B). IMD administration also attenuated
   cardiac fibrosis induced by fructose-drinking in rats, as shown by a
   decreased cardiac interstitial and perivascular fibrosis area and
   down-regulated mRNA and protein levels of pro-fibrotic gene collagen
   type I alpha 1 chain (Col1a1) and collagen type III alpha 1 chain
   (Col3a1) ([113]Figure 1C–E). Moreover, IMD administration significantly
   decreased FBG, fasting serum insulin levels, insulin resistance, serum
   triglycerides, and LDL-C levels, and it increased the serum HDL-C
   levels of rats ([114]Table S1). These findings demonstrated that
   exogenous IMD mitigates DCM induced by fructose drinking.

Figure 1.

   [115]Figure 1

   [116]Figure 1
   [117]Open in a new tab

   Exogenous IMD alleviates DCM in rats. (A) Hematoxylin–eosin staining of
   representative heart sections and cardiomyocyte cross-sectional area
   quantification in rats. Scale bar: 50 μm. n = 4. (B) Quantitative
   real-time PCR analysis of Nppa and Nppb mRNA expression in the hearts
   of diabetic rats. n = 3–4. (C) Sirius red staining of myocardial
   interstitial and perivascular fibrosis area with representative images
   and quantification in rats. Scale bar: 50 μm. n = 3. (D) Quantitative
   real-time PCR analysis of Col1a1 and Col3a1 mRNA expression in DCM rat
   hearts. n = 3–4. (E) Western blot analysis of Col1a1 and Col3a1 protein
   levels in DCM rat hearts. n = 3. (F) Representative images and
   quantification of surface (μm^2) in NRCMs analyzed by ImageJ 1.53k.
   Scale bar: 100 μm, 50 μm. n = 3–6. (G) Quantitative real-time PCR
   analysis of Nppa and Nppb mRNA expression in NRCMs. n = 3–4. (H)
   Representative images and quantification of immunofluorescence staining
   for αSMA (green), Col1a1 (red) and DAPI (blue) in primary cultured rat
   cardiac fibroblasts. Merged images are shown. Scale bar: 50 μm. n = 3.
   Data are mean ± SD, * p < 0.05, ** p < 0.01.

   In vitro, we investigated how IMD influenced PA-induced cardiomyocyte
   hypertrophy and cardiac fibroblast activation in rats. It was shown
   that IMD treatment reduced cell surface area and Nppa and Nppb mRNA
   levels of NRCMs treated with PA ([118]Figure 1F,G). IMD treatment also
   down-regulated α-SMA and Col1a1 levels of rat cardiac fibroblast
   treated with PA ([119]Figure 1H). These results suggested that IMD
   administration alleviated PA-induced cardiomyocyte hypertrophy and
   cardiac fibroblast activation in rats.

2.3. IMD Overexpression Alleviates DCM in Mice

   Given the findings that exogenous IMD alleviates DCM, we next explored
   whether endogenous IMD has beneficial effects on DCM of IMD-transgenic
   (IMDtg) mice induced by an HFD. Firstly, quantitative real-time PCR
   results indicated that IMD was significantly overexpressed in IMDtg
   mice hearts ([120]Figure S3A). Next, we assessed cardiac function and
   cardiac pathological morphology in each group of mice. IMD
   overexpression reduced the heart weight/tibial length (HW/TL) ratio,
   whereas the heart weight/body weight (HW/BW) ratio had no statistically
   significant changes ([121]Figure 2A). Echocardiography and Doppler
   echocardiography showed that IMD overexpression decreased the LV wall
   thickness and increased LV EF, fractional shortening (FS), and the E/A
   ratio in IMDtg mice. These results suggested that IMD overexpression
   mitigated induced cardiac systolic and diastolic dysfunction in HFD-fed
   mice ([122]Figure 2B,C and [123]Table S2). Meanwhile, we found that IMD
   overexpression also attenuated cardiac hypertrophy, fibrosis, and lipid
   accumulation in IMDtg mice myocardium, as evidenced by the reduction in
   the myocardial cross-sectional area, pro-hypertrophic gene expression,
   interstitial and perivascular fibrosis area, Oil Red O positive area,
   and pro-fibrotic gene expression ([124]Figure 2D–I). The prevalence of
   hypertension in T2DM individuals was up to three times higher than in
   non-diabetic individuals [[125]43,[126]44]. IMD also improved
   hypertension in HFD-fed IMDtg mice ([127]Table S3). Furthermore, IMD
   overexpression significantly decreased fasting serum insulin levels,
   insulin resistance, serum levels of triglycerides, total cholesterol,
   LDL-C, and increased serum HDL-C levels in HFD-fed IMDtg mice versus
   HFD-fed WT mice ([128]Table S4). These data indicated that endogenous
   IMD mitigated DCM induced by an HFD.

Figure 2.

   [129]Figure 2
   [130]Open in a new tab

   IMD overexpression alleviates DCM in mice. (A) Representative images of
   hearts, HW/BW ratio, and HW/TL ratio of diabetic WT and IMDtg mice.
   Scale bar: 10 mm. n = 6–7. (B) Representative echocardiographic images
   of diabetic WT and IMDtg mice. (C) Representative pulsed Doppler
   echocardiography pictures, early diastolic mitral flow velocity (E),
   late diastolic mitral flow velocity (A), and relative quantification of
   the mitral E/A ratio of diabetic WT and IMDtg mice. n = 4–6. (D)
   Hematoxylin–eosin staining of representative heart sections and
   cardiomyocyte cross-sectional area quantification in mice. Scale bar:
   50 μm. n = 4. (E) Quantitative real-time PCR analysis of Nppa and Nppb
   mRNA expression in the hearts of diabetic WT and IMDtg mice. n = 3–4.
   (F) Sirius red staining of myocardial interstitial and perivascular
   fibrosis area with representative images and quantification from
   different mice. Scale bar: 50 μm. n = 3. (G) Oil Red O staining of
   myocardial interstitium with representative images and quantification
   from different mice. Scale bar: 50 μm. n = 5. (H) Quantitative
   real-time PCR analysis of Col1a1 and Col3a1 mRNA expression in the
   hearts of diabetic WT and IMDtg mice. n = 4. (I) Western blot analysis
   of Col1a1 and Col3a1 protein levels in the hearts of diabetic WT and
   IMDtg mice. n = 3. Data are mean ± SD, * p < 0.05, ** p < 0.01. ns: no
   significant difference.

2.4. IMD Deficiency Exacerbates DCM in Mice

   To further confirm the cardiac protective effects of endogenous IMD on
   DCM, HFD-fed, IMD-knockout (IMD^−/−) mice were used. Firstly,
   quantitative real-time PCR results indicated that IMD was strongly
   down-regulated in IMD^−/− mice hearts ([131]Figure S3B). Then, we
   assessed cardiac function and cardiac pathological morphology in each
   group of mice. IMD deficiency increased the HW/BW ratio and HW/TL ratio
   in HFD-fed, IMD^−/− mice versus HFD-fed WT mice ([132]Figure 3A).
   Echocardiography revealed that IMD deficiency significantly exacerbated
   cardiac systolic and diastolic dysfunction in HFD-fed, IMD^−/− mice
   ([133]Figure 3B,C and [134]Table S5). Consistent with this, the results
   of hematoxylin and eosin (H&E) staining, quantitative real-time PCR,
   and Sirius red and Oil Red O staining confirmed that IMD deficiency
   effectively aggravated cardiac hypertrophy, fibrosis, and lipid
   accumulation in the myocardium of HFD-fed, IMD^−/− mice ([135]Figure
   3D–I). IMD deficiency further exacerbated the hypertension of HFD-fed,
   IMD^−/− mice ([136]Table S6). Moreover, IMD deficiency increased FBG,
   fasting serum insulin levels, insulin resistance, serum levels of
   triglycerides, and total cholesterol in HFD-fed, IMD^−/− mice versus
   HFD-fed WT mice ([137]Table S7). Taken together, these results
   suggested that endogenous IMD deficiency aggravated the HFD-induced DCM
   of IMD^−/− mice.

Figure 3.

   [138]Figure 3
   [139]Open in a new tab

   IMD deficiency exacerbates DCM in mice. (A) Representative images of
   hearts, the HW/BW ratio, and HW/TL ratio of diabetic WT and IMD^−/−
   mice. Scale bar: 10 mm. n = 6–8. (B) Representative echocardiographic
   images of diabetic WT and IMD^−/− mice. (C) Representative pulsed
   Doppler echocardiography pictures, early diastolic mitral flow velocity
   (E), late diastolic mitral flow velocity (A), and relative
   quantification of the mitral E/A ratio of diabetic WT and IMD^−/− mice.
   n = 4–5. (D) Hematoxylin–eosin staining of representative heart
   sections and cardiomyocyte cross-sectional area quantification in mice.
   Scale bar: 50 μm. n = 4. (E) Quantitative real-time PCR analysis of
   Nppa and Nppb mRNA expression in the hearts of diabetic WT and IMD^−/−
   mice. n = 3–4. (F) Sirius red staining of myocardial interstitial and
   perivascular fibrosis area with representative images and
   quantification in mice. Scale bar: 50 μm. n = 3. (G) Oil red O staining
   of myocardial interstitium with representative images and
   quantification from different mice. Scale bar: 50 μm. n = 5. (H)
   Quantitative real-time PCR analysis of Col1a1 and Col3a1 mRNA
   expression in the hearts of diabetic WT and IMD^−/− mice. n = 3–4. (I)
   Western blot analysis of Col1a1 and Col3a1 protein levels in the hearts
   of diabetic WT and IMD^−/− mice. n = 3. Data are mean ± SD, * p < 0.05,
   ** p < 0.01. ns: no significant difference.

2.5. IMD Alleviates DCM by Up-Regulating CPT-1β

   CPT-1β is the rate-limiting enzyme responsible for β-oxidation of
   long-chain fatty acids
   [[140]14,[141]15,[142]16,[143]17,[144]18,[145]19]. To explore whether
   IMD alleviated DCM depended on the role of CPT-1β; we firstly performed
   RNA sequencing in the hearts of HFD-fed WT mice. HFD-fed WT mice showed
   496 up-regulated genes and 367 down-regulated genes (with at least a
   two-fold change) and significant enrichment in fatty acid metabolism
   pathways ([146]Figure 4A,B). The heat map revealed the up-regulation of
   Cpt1b in HFD-fed WT mice ([147]Figure 4C). Real-time PCR also revealed
   the up-regulation of the Cpt1b mRNA level in HFD-fed WT mice and in the
   hearts of fructose-drinking rats ([148]Figure 4D). These results
   indicated that CPT-1β was a key regulator involved in DCM. Then, we
   performed RNA sequencing on hearts from HFD-fed, IMD^−/− mice and WT
   mice to explore whether IMD alleviated DCM by regulating CPT-1β. It was
   found that HFD-fed, IMD^−/− mice showed 1397 up-regulated genes and 893
   down-regulated genes versus HFD-fed WT mice (with at least a two-fold
   change) and significant enrichment in fatty acid metabolism pathways
   ([149]Figure 4E,F). The heat map and real-time PCR showed the
   down-regulation of Cpt1b mRNA expression in HFD-fed, IMD^−/− mice
   ([150]Figure 4G,H). Thus, CPT-1β might be a crucial target for IMD to
   protect against DCM. Then, we performed RNA sequencing on hearts from
   HFD-fed, IMDtg mice and WT mice to confirm whether CPT-1β was a key
   target of IMD. The heat map and real-time PCR showed the up-regulation
   of Cpt1b mRNA expression in HFD-fed, IMDtg mice versus HFD-fed WT mice
   ([151]Figure 4I,J). Therefore, IMD could up-regulate Cpt1b mRNA
   expression in the myocardium of mice. Then, we further verified whether
   IMD played a protective role in DCM by up-regulating CPT-1β. CPT-1β is
   the main isoform in the heart [[152]14,[153]16]. We found that the mRNA
   level of Cpt1b was higher than Cpt1a in NRCM ([154]Figure 5A). The
   enzyme activity of CPT-1β and content of acetyl-CoA generated by fatty
   acid β-oxidation in plasma were significantly increased in T2DM
   patients ([155]Figure 5B,C). Fructose-drinking rats showed increased
   enzyme activity, mRNA, protein levels of CPT-1β, and content of
   acetyl-CoA, and these levels were further enhanced by IMD
   administration ([156]Figure 5D–G). Moreover, IMD overexpression
   elevated the enzyme activity and protein level of CPT-1β and the
   content of acetyl-CoA in HFD-fed, IMDtg mice ([157]Figure 5H–J),
   whereas IMD deficiency decreased the above phenomena ([158]Figure
   5K–M).

Figure 4.

   [159]Figure 4
   [160]Open in a new tab

   IMD up-regulates CPT-1β. RNA-sequencing analysis of hearts in mice. (A)
   Volcano plot showing the transcript expression profiles of
   differentially expressed genes in the hearts of the WT diabetic mice,
   respectively. n = 3. (B) KEGG pathway enrichment analysis showing the
   20 most significantly enriched signaling pathways for differentially
   expressed genes in the hearts of WT diabetic mice. n = 3. (C) Heat map
   of the microarray results showing the 36 (ranked by p-values)
   differentially expressed fatty acid metabolism genes in hearts from the
   WT diabetic mice. Red, up-regulated; blue, down-regulated; white, no
   change. n = 3. (D) Quantitative real-time PCR analysis of Cpt1b mRNA
   expression in the hearts of WT diabetic mice and diabetic rats. n = 4.
   (E) Volcano plot showing the transcript expression profiles of
   differentially expressed genes in the hearts from the diabetic WT and
   IMD^−/− mice, respectively. n = 3. (F) KEGG pathway enrichment analysis
   showing the 20 most significantly enriched signaling pathways for
   differentially expressed genes in the hearts of diabetic WT and IMD^−/−
   mice. n = 3. (G) Heat map of the microarray results showing the 28
   (ranked by p-values) differentially expressed fatty acid metabolism
   genes in hearts from the diabetic WT and IMD^−/− mice. Red,
   up-regulated; blue, down-regulated; white, no change. n = 3. (H)
   Quantitative real-time PCR analysis of Cpt1b mRNA expression in the
   hearts of diabetic WT and IMD^−/− mice. n = 4. (I) Heat map of the
   microarray results showing the 23 (ranked by P-values) differentially
   expressed fatty acid metabolism genes in hearts from the diabetic WT
   and IMDtg mice. Red, up-regulated; blue, down-regulated; white, no
   change. n = 3. (J) Quantitative real-time PCR analysis of Cpt1b mRNA
   expression in the hearts of diabetic WT and IMDtg mice. n = 4. Data are
   mean ± SD, ** p < 0.01.

Figure 5.

   [161]Figure 5

   [162]Figure 5
   [163]Open in a new tab

   IMD alleviates DCM by up-regulating CPT-1β. (A) Quantitative real-time
   PCR analysis of Cpt1a and Cpt1b mRNA expression in NRCMs. n =3. (B)
   Enzyme-linked immunosorbent assay of plasma CPT-1β activity in T2DM
   patients and healthy controls. n = 38. (C) Enzyme-linked immunosorbent
   assay of plasma acetyl-CoA concentration in T2DM patients and healthy
   controls. n = 38–41. (D) Enzyme-linked immunosorbent assay of serum
   CPT-1β activity in diabetic rats. n = 6. (E) Quantitative real-time PCR
   analysis of Cpt1b mRNA expression in the hearts of diabetic rats. n =
   4. (F) Western blot analysis of CPT-1β protein levels in diabetic rat
   hearts. n = 3. (G) Enzyme-linked immunosorbent assay of serum
   acetyl-CoA concentration in diabetic rats. n = 5–6. (H) Enzyme-linked
   immunosorbent assay of serum CPT-1β activity of diabetic WT and IMDtg
   mice. n = 6. (I) Western blot analysis of CPT-1β protein levels in the
   hearts of diabetic WT and IMDtg mice. n = 3. (J) Enzyme-linked
   immunosorbent assay of serum acetyl-CoA concentration in diabetic WT
   and IMDtg mice. n = 5–6. (K) Enzyme-linked immunosorbent assay of serum
   CPT-1β activity of diabetic WT and IMD^−/− mice. n = 6. (L) Western
   blot analysis of CPT-1β protein levels in the hearts of diabetic WT and
   IMD^−/− mice. n = 3. (M) Enzyme-linked immunosorbent assay of serum
   acetyl-CoA concentration in diabetic WT and IMD^−/− mice. n = 6. (N)
   Enzyme-linked immunosorbent assay of NRCM supernatant CPT-1β activity.
   n = 5–6. (O) Quantitative real-time PCR analysis of Cpt1b mRNA
   expression in NRCMs. n = 3–4. (P) Western blot analysis of CPT-1β
   protein levels in NRCMs. n = 3. (Q) Enzyme-linked immunosorbent assay
   of acetyl-CoA concentration in supernatant of NRCMs. n = 4. (R)
   Representative images treated with CPT-1β siRNA and quantification of
   surface (μm^2) in NRCMs analyzed by ImageJ 1.53k. Scale bar: 100 μm, 50
   μm. n = 3–4. (S) Quantitative real-time PCR analysis of Nppa and Nppb
   mRNA expression in NRCMs treated with CPT-1β siRNA. n = 4. (T)
   Representative images and quantification of immunofluorescence staining
   for αSMA (green), Col1a1 (red) and DAPI (blue) in primary cultured rat
   cardiac fibroblasts. Merged images are shown. Scale bar: 50 μm. n = 3.
   (U) Enzyme-linked immunosorbent assay of acetyl-CoA concentration in
   supernatant of NRCMs. n = 4–6. Data are mean ± SD, * p < 0.05, ** p <
   0.01. ns: no significant difference.

   Similar results were observed in vitro. IMD treatment increased enzyme
   activity, mRNA, protein levels of CPT-1β, and content of acetyl-CoA in
   PA-treated NRCMs ([164]Figure 5N–Q). To verify that IMD alleviated DCM
   via up-regulating CPT-1β, small-interfering RNA (siRNA) of CPT-1β was
   used to knock down CPT-1β expression in NRCMs. CPT-1β silencing
   ([165]Figure S4A,B) abolished the effects of IMD in alleviating
   cardiomyocyte hypertrophy and cardiac fibroblast activation and
   increasing acetyl-CoA content in NRCMs ([166]Figure 5R–U). These
   results suggested that IMD alleviated cardiomyocyte hypertrophy and
   cardiac fibroblast activation in rats by up-regulating CPT-1β.

2.6. IMD Up-Regulates CPT-1β via the PI3K/Akt Signaling Pathway

   IMD binds to its receptor complexes, CRLR/RAMPs, to exert biological
   effects [[167]30,[168]31,[169]32,[170]33,[171]45]. Firstly, to explore
   whether IMD alleviated DCM by up-regulating CPT-1β through its receptor
   complexes, IMD receptor antagonist IMD[17–47] was used. Preincubation
   with IMD[17–47] blocked the effects of IMD on up-regulating enzyme
   activity, mRNA, and protein levels of CPT-1β in NRCMs treated with PA
   ([172]Figure 6A–C). Pretreatment with IMD[17–47] also blocked the
   effects of IMD on inhibiting cardiomyocyte hypertrophy ([173]Figure
   6D,E) and cardiac fibroblast activation ([174]Figure 6F) and increasing
   acetyl-CoA content ([175]Figure 6G) in PA-treated NRCMs. These results
   suggested that IMD alleviated cardiomyocyte hypertrophy and cardiac
   fibroblast activation and increased acetyl-CoA content by up-regulating
   CPT-1β through its receptor complexes.

Figure 6.

   [176]Figure 6
   [177]Open in a new tab

   IMD up-regulates CPT-1β via its receptor complex. (A) Enzyme-linked
   immunosorbent assay of NRCM supernatant CPT-1β activity. n = 5–6. (B)
   Quantitative real-time PCR analysis of Cpt1b mRNA expression in NRCMs.
   n = 3–4. (C) Western blot analysis of CPT-1β protein levels in NRCMs. n
   = 3. (D) Representative images treated with IMD[17–47] and
   quantification of surface (μm^2) in NRCMs analyzed by ImageJ 1.53k.
   Scale bar: 100 μm, 50 μm. n = 3–5. (E) Quantitative real-time PCR
   analysis of Nppa and Nppb mRNA expression in NRCMs. n = 3–4. (F)
   Representative images and quantification of immunofluorescence staining
   for αSMA (green), Col1a1 (red) and DAPI (blue) in primary cultured rat
   cardiac fibroblasts. Merged images are shown. Scale bar: 50 μm. n = 3.
   (G) Enzyme-linked immunosorbent assay of acetyl-CoA concentration in
   the supernatants of NRCMs. n = 4. +: added. −: not added. Data are mean
   ± SD, * p < 0.05, ** p < 0.01.

   The binding of IMD to its receptor complexes leads to the activation of
   post-receptor signaling pathways, such as cyclic adenosine
   monophosphate/protein kinase A (cAMP/PKA), AMP-activated protein kinase
   (AMPK), and PI3K/Akt pathways [[178]30,[179]31,[180]32]. In this study,
   we found that the phosphorylation of AMPK and Akt was apparently
   elevated in PA-treated NRCMs and heart tissues of fructose-drinking
   rats, which were further enhanced by IMD administration, whereas IMD
   treatment had no significant effect on the phosphorylation of PKA in
   PA-treated NRCMs ([181]Figure 7A–C). IMD overexpression elevated the
   phosphorylation of AMPK and Akt in the heart tissues of HFD-fed, IMDtg
   mice, whereas IMD deficiency decreased the above phenomena ([182]Figure
   7D,E). These findings indicated that AMPK and PI3K/Akt signaling
   pathways were activated by IMD in DCM and hypertrophic cardiomyocytes.
   To verify whether AMPK and PI3K/Akt mediated the effects of IMD on
   up-regulating CPT-1β, AMPK inhibitor Compound C and PI3K inhibitor
   LY294002 were used. Only LY294002 pretreatment blocked the effects of
   IMD on up-regulating enzyme activity and the protein level of CPT-1β
   and the content of acetyl-CoA in NRCMs ([183]Figure 7F–I), although
   both Compound C and LY294002 reversed the effects of IMD on alleviating
   cardiomyocyte hypertrophy and cardiac fibroblast activation in rats
   ([184]Figure 7J–L). These results indicated that IMD up-regulated
   CPT-1β, then inhibited cardiomyocyte hypertrophy and cardiac fibroblast
   activation, and increased the content of the acetyl-CoA via PI3K/Akt
   signaling pathway.

Figure 7.

   [185]Figure 7

   [186]Figure 7
   [187]Open in a new tab

   IMD up-regulates CPT-1β via the PI3K/Akt signaling pathway. (A) Western
   blot analysis of p-AMPK (Thr172), AMPK, p-Akt (Ser473), Akt, p-PKA
   (Thr197), and PKA protein levels in NRCMs. n = 3. (B) Western blot
   analysis of p-AMPK (Thr172) and AMPK protein levels in DCM rat hearts.
   n = 3. (C) Western blot analysis of p-Akt (Ser473) and Akt protein
   levels in DCM rat hearts. n = 3. (D) Western blot analysis of p-AMPK
   (Thr172) and AMPK protein levels in the hearts of diabetic WT, IMDtg,
   and IMD^−/− mice. n = 3. (E) Western blot analysis of p-Akt (Ser473)
   and Akt protein levels in the hearts of diabetic WT, IMDtg, and IMD^−/−
   mice. n = 3. (F) Enzyme-linked immunosorbent assay of NRCM supernatant
   CPT-1β activity. n = 5–6. (G,H) Western blot analysis of CPT-1β protein
   levels in NRCMs. n = 3. (I) Enzyme-linked immunosorbent assay of
   acetyl-CoA concentration in the supernatants of NRCMs. n = 4. (J)
   Representative images and quantification of surface (μm^2) in NRCMs
   analyzed by ImageJ 1.53k. Scale bar: 100 μm, 50 μm. n = 3–4. (K)
   Quantitative real-time PCR analysis of Nppa and Nppb mRNA expression in
   NRCMs. n = 4. (L) Representative images and quantification of
   immunofluorescence staining for αSMA (green), Col1a1 (red) and DAPI
   (blue) in primary cultured rat cardiac fibroblasts. Merged images are
   shown. Scale bar: 50 μm. n = 3. +: added. −: not added. Data are mean ±
   SD, * p < 0.05, ** p < 0.01. ns: no significant difference.

3. Discussion

   In this study, we highlighted the beneficial effect of IMD on DCM and
   its underlying mechanisms. Specifically, this study innovatively
   demonstrated that (a) the levels of IMD and its receptor complexes were
   reduced in the heart tissues of DCM rats and mice. (b) Exogenous and
   endogenous IMD mitigated cardiac hypertrophy, fibrosis, dysfunction,
   and lipid accumulation in DCM rats and IMD-transgenic DCM mice, whereas
   knockout of IMD worsened these pathological processes in IMD-knockout
   DCM mice. (c) CPT-1β enzyme activity, mRNA and protein levels, and
   acetyl-CoA content were increased in T2DM patients, rats and mice. IMD
   increased enzyme activity, mRNA, protein levels of CPT-1β, and the
   content of acetyl-CoA in the serum and cardiac tissues of DCM rats. IMD
   overexpression elevated the enzyme activity and protein level of CPT-1β
   and the content of acetyl-CoA in serum and cardiac tissues of IMDtg DCM
   mice. Knockdown of CPT-1β blocked the effects of IMD on increasing the
   acetyl-CoA content in PA-treated NRCMs and on inhibiting cardiomyocyte
   hypertrophy and cardiac fibroblast activation. IMD receptor antagonist
   IMD[17–47] and PI3K/Akt inhibitor LY294002 reversed the effects of IMD
   on up-regulating enzyme activity, mRNA, and protein level of CPT-1β and
   on the content of acetyl-CoA and on inhibiting cardiomyocyte
   hypertrophy and cardiac fibroblast activation. We revealed that IMD
   alleviates DCM by up-regulating CPT-1β via CRLR/RAMP receptor complexes
   and PI3K/Akt signaling. Therefore, targeting the restoration of the IMD
   level may be a potential clinical therapeutic target for DCM treatment.

   Our study revealed the protective role of IMD in DCM. To explore the
   effect of IMD on DCM, rats and wildtype, IMD^−/−, and IMDtg mice were
   used [[188]8,[189]39,[190]46,[191]47,[192]48,[193]49,[194]50,[195]51]
   to induce DCM under different pathologies. We did not observe any
   abnormalities in IMDtg mice and IMD^−/− mice, which are consistent with
   previous studies [[196]34,[197]40,[198]41,[199]51,[200]52]. In this
   study, IMDtg mice and IMD^−/− mice did not have cardiac hypertrophy,
   fibrosis, dysfunction, hypertension, and abnormal glucose and lipid
   metabolism, which were indistinguishable from their WT littermates. We
   did not observe other systems or organs of IMDtg mice and IMD^−/− mice;
   this is a limitation. In other studies, IMD^−/− mice exhibited a
   primary immunosuppression phenotype, impaired vessel hierarchical
   structure, reduced blood perfusion, vascular leakage, and increased
   inflammation [[201]53,[202]54,[203]55,[204]56]. Therefore, IMD plays an
   important role in vascular structure, vascular development, and
   inflammatory immunity.

   According to previous studies, with minor modifications
   [[205]57,[206]58], drinking 10% fructose water for 12 weeks resulted in
   insulin resistance, cardiac hypertrophy, and fibrosis in SD rats.
   HFD-fed resulted in insulin resistance, cardiac hypertrophy, fibrosis
   and diastolic and systolic dysfunction and lipid accumulation in mice
   [[207]46,[208]47,[209]48,[210]49,[211]50,[212]51].

   Endogenous IMD is found in the brain, pituitary, heart, kidney,
   gastrointestinal tract, plasma, pancreas, lung, spleen, thymus, and
   ovary [[213]33]. Endogenous IMD is highly expressed in the heart, which
   can secrete from cardiomyocytes and cardiac fibroblasts
   [[214]30,[215]31,[216]32,[217]33,[218]34,[219]36,[220]59]. In this
   study, we did not detect the expression of IMD in cardiac fibroblasts,
   which is a limitation. In this study, we found that cardiac mRNA levels
   of IMD, mRNA, and protein levels of its receptor complexes, CRLR/RAMPs,
   were reduced in DCM rats and mice, which is consistent with previous
   studies [[221]60]. Other studies reported that IMD expression decreased
   in the plasma, cardiac tissue, adipose tissue, and aortic root
   atherosclerotic lesions of db/db mice, HFD-induced obese mice,
   hyperhomocysteinemia mice, HFD-fed ApoE-/- mice, and diabetic rats
   [[222]40,[223]41,[224]42,[225]51,[226]60]. CGRP superfamily members ADM
   and CGRP are down-regulated in the myocardium of diabetic rats
   [[227]61,[228]62]. We also detected that mRNA levels of IMD were
   down-regulated in hypertrophic cardiomyocytes. These data may indicate
   that IMD acts in an autocrine or paracrine manner [[229]40]. T2DM
   characterized with hyperglycemia due to impaired insulin secretion
   and/or action may lead to the down-regulation of IMD, indicating that
   IMD is involved in DCM [[230]2]. Hirose et al. reported that the
   expression of AM2/IMD is enhanced in the failing heart, and
   up-regulated AM2/IMD in the failing heart seems to be related to its
   cardio-protective effects [[231]38,[232]63]. In this study, although
   IMD is down-regulated in DCM, exogenous IMD administration protected
   against DCM in T2DM rats. These differences might be attributed to
   different disease states and stages leading to different expression
   levels of IMD. We found that CPT-1β enzyme activity, mRNA, and protein
   levels were increased in T2DM patients, rats and mice, which is in line
   with Nakamura’s report [[233]17]. These results suggested that
   decreased endogenous IMD led to compensatory up-regulation of CPT-1β.

   Then, we observed that both exogenous IMD administration and IMD
   overexpression mitigated cardiac hypertrophy, fibrosis, diastolic and
   systolic dysfunction, and lipid accumulation in T2DM rats and mice. IMD
   alleviated cardiomyocyte hypertrophy and cardiac fibroblast activation
   in rats induced by PA. Conversely, IMD deficiency exacerbated the
   development of DCM in T2DM mice. These data revealed that IMD was
   essential for alleviating DCM. In line with previous studies
   [[234]30,[235]34,[236]35,[237]36,[238]37,[239]38,[240]40,[241]41,[242]5
   9], IMD can improve many cardiovascular diseases such as diabetic
   ischemic heart injury, cardiac hypertrophy, cardiac fibrosis,
   congestive heart failure, and hypertension. IMD also can improve
   metabolic syndromes such as obesity and insulin resistance in T1DM
   rats, T2DM mice, and ApoE^−/− atherosclerosis mice
   [[243]39,[244]40,[245]41,[246]42].

   CPT-1β is the rate-limiting enzyme responsible for β-oxidation of
   long-chain fatty acids
   [[247]14,[248]15,[249]16,[250]17,[251]18,[252]19]. Overexpression of
   CPT-1 can enhance fatty acid oxidation, reduce lipid accumulation, and
   improve insulin sensitivity in HFD-fed rats [[253]18]. Deletion or
   inhibition of CPT-1β can lead to the inhibition of fatty acid
   oxidation, myocardial lipid accumulation, impaired insulin sensitivity,
   heart regeneration, cardiac hypertrophy, cardiac fibrosis, and cardiac
   dysfunction in mice [[254]14,[255]19,[256]20,[257]21,[258]22,[259]23].
   Moreover, many studies have reported that multiple bioactive molecules
   regulate CPT-1β expression [[260]24,[261]25,[262]26,[263]27]. In this
   study, we found that IMD up-regulated the CPT-1β mRNA level, protein
   level and enzyme activity, and acetyl-CoA content in T2DM rats, T2DM
   mice, and hypertrophic cardiomyocytes. Then, CPT-1β knockdown prevented
   the effects of IMD on inhibiting cardiomyocyte hypertrophy, cardiac
   fibroblast activation, and up-regulating acetyl-CoA content. Thus,
   these findings revealed that IMD may protect against DCM by
   up-regulating CPT-1β expression.

   IMD binds to its receptor complexes, CRLR/RAMPs, and activates
   post-receptor signaling pathways such as cAMP/PKA, AMPK, and PI3K/Akt
   to exert biological effects [[264]30,[265]31,[266]32]. In this work, we
   found that the phosphorylation of AMPK and Akt was apparently elevated
   in cardiac tissues of DCM rats and mice and in hypertrophic
   cardiomyocytes, which were further enhanced after exogenous IMD
   administration and IMD overexpression. Only PI3K inhibitor LY294002
   pretreatment blocked the effects of IMD on up-regulating CPT-1β enzyme
   activity and protein levels and acetyl-CoA content in NRCMs. These
   results indicated that IMD up-regulated CPT-1β and then inhibited
   PA-induced rat cardiomyocyte hypertrophy and cardiac fibroblast
   activation and increased the NRCM content of acetyl-CoA via the
   PI3K/Akt signaling pathway. These phenomena were in line with previous
   studies [[267]64,[268]65,[269]66,[270]67,[271]68]. However, our results
   showed that the inhibition of AMPK did not block the up-regulatory
   effect of IMD on CPT-1β. In this study, we found that IMD alleviates
   DCM through activation of the PI3K/Akt signaling pathway. PI3K/Akt can
   be activated by phosphorylation of the insulin receptor substrate after
   insulin binding to its receptor [[272]10,[273]64,[274]69]. Insulin can
   alleviate DCM by binding to its receptor located at the plasma membrane
   and then stimulating the uptake of glucose into cardiac muscle
   [[275]10,[276]69,[277]70,[278]71,[279]72,[280]73,[281]74]. In this
   study, we found that fasting serum insulin levels increased, and
   insulin resistance was observed in T2DM rats and mice, which are
   consistent with previous studies [[282]10,[283]51,[284]70]. Tissue
   resistance to the action of insulin constitutes the hallmark of T2DM,
   promoting reduced insulin sensitivity and compensatory increase in
   insulin [[285]10,[286]51,[287]70]. It was reported that IMD can protect
   against diabetic ischemic heart injury in rats [[288]59]. CGRP
   superfamily members ADM and CGRP can also improve cardiac dysfunction
   in diabetic rats [[289]66,[290]75]. In this study, we found that IMD
   can alleviate DCM by up-regulating CPT-1β through activation of the
   PI3K/Akt signaling pathway, which is consistent with previous studies
   [[291]59,[292]66,[293]75]. IMD might also alleviate DCM partly through
   improving insulin resistance in T2DM rats and mice.

4. Materials and Methods

4.1. Human Subjects

   Between January 2017 and December 2017, male participants with type 2
   diabetes mellitus (n = 38) and without diabetes (n = 41) were included
   in this study. Inclusion criteria and exclusion criteria were
   previously described [[294]51]. Informed consent was obtained from all
   participants for this study. The study on humans was conducted
   according to the ethical guidelines of the 1975 Declaration of
   Helsinki. The protocol was approved by the Ethics Committee of Peking
   University International Hospital (No. 2016-064).

4.2. Animals

   All animal care and experimental protocols followed the Guide for the
   Care and Use of Laboratory Animals published by the US National
   Institutes of Health (NIH Publication, 8th Edition, 2011). The Animal
   Care Committee of Peking University Health Science Center (Beijing,
   China) approved all animal experimental procedures (No. LA2020042). All
   experiments complied with international guidelines on the ethical use
   of animals.

   Sprague Dawley (SD) rats and Neonatal SD rats (1–3-day-old) were
   purchased from the Animal Center, Peking University Health Science
   Center (No. LA2020042). The Model Animal Research Center of Nanjing
   University (Nanjing, China) provided IMDtg mice, IMD^−/− mice, and
   their WT littermates as controls with C57BL/6 background, as described
   before [[295]59,[296]76].

4.3. DCM Models of Rats and Mice

   Male SD rats (8 weeks old) were randomly assigned to 3 groups: (1)
   control (CON) group—rats were given normal drinking water for 12 weeks;
   (2) fructose-drinking rat (FDR) group—rats were given 10% fructose
   drinking water for 12 weeks; and (3) FDR plus IMD (FDR + IMD)
   group—rats were given 10% fructose drinking water for 12 weeks and 4
   weeks of subcutaneous infusion of IMD at 100 ng/kg/h via an Alzet
   Mini-osmotic Pump [[297]57,[298]58] with minor modifications. All rats
   were fasted overnight and killed by exsanguination. The hearts were
   rapidly removed for further studies.

   Male IMD^−/−, IMDtg, and WT littermate mice (8–10 weeks old) were
   randomly assigned to 2 groups: (1) CON group—mice received a normal
   diet for 16 weeks and (2) HFD group—mice were fed with HFD (60% kcal
   from lard, D12492) for 16 weeks [[299]46,[300]47,[301]48]. After
   echocardiography and hemodynamic measurements, all mice were fasted
   overnight and killed by exsanguination. The hearts were rapidly removed
   for further studies.

4.4. Echocardiography

   Mice were subjected to echocardiography using a Vevo 770 T M Imaging
   System (Visual Sonics, Toronto, ON, Canada) and a 30 MHz probe. We used
   2-D-directed M-mode from the LV short axis to record the LV internal
   diameter in systole (LVID; s) or in diastole (LVID; d), LV posterior
   wall thickness in systole (LVPW; s) or in diastole (LVPW; d), LV
   anterior wall thickness in systole (LVAW; s) or in diastole (LVAW; d),
   LV volume in systole (LV Vol; s) or in diastole (LV Vol; d), LV EF, and
   FS. Early diastolic mitral flow velocity (E)/late diastolic mitral flow
   velocity (A) was evaluated by pulse Doppler ultrasound. Data from 5
   consecutive cardiac cycles were averaged.

4.5. Blood Pressure Measurement

   After echocardiography, the non-invasive computerized tail-cuff system
   (BP-98A, Softron, Tokyo, Japan) was used to measure the systolic blood
   pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure
   (MBP) of the mice. A thermostat was used to keep the body temperature
   of the mice constant before blood pressure was measured (30 °C–33 °C).
   The blood pressure of the mice was monitored continuously until the
   blood pressure of the mice stabilized. Data from 5 individual
   measurements were averaged [[302]34].

4.6. Neonatal Rat Cardiomyocyte (NRCM) and Cardiac Fibroblast Culture

   We isolated ventricles from neonatal SD rats (1–3 days old), shredded
   and digested them, and collected the supernatant. The cell suspension
   was filtered and pre-plated in two 25-mm^2 culture flasks for 90 min at
   37 °C to allow the fibroblasts to adhere to the flasks. The
   non-attached cardiomyocytes were plated in several 6-well cell culture
   plates. Cardiomyocytes exhibited a rhombic, triangular, or polygonal
   shape under the microscope. The cardiomyocytes formed beating islands.
   Fibroblasts were spindle-shaped or star-like ([303]Figure S5A).
   Fibroblast purity was assessed by staining with Vimentin ([304]Figure
   S5B).

   Then, 300 μmol/L PA was selected as the optimal concentration and 24 h
   was selected as the optimal stimulation time based on the effect on
   cell viability ([305]Figure S6A–D) [[306]77,[307]78]. The
   cardiomyocytes and fibroblasts were starved overnight with a 1% fetal
   bovine serum medium. After incubation with IMD (1 × 10^−7 mol/L;
   Phoenix Pharmaceuticals, Belmont, CA, USA) for 30 min, the
   cardiomyocytes and fibroblasts were stimulated with PA (300 μmol/L) for
   24 h to induce cardiomyocyte hypertrophy and fibroblast activation
   [[308]34,[309]35,[310]77,[311]78,[312]79,[313]80]. To explore the role
   of CRLR/RAMPs and their signaling pathway, the cardiomyocytes and
   fibroblasts were pre-incubated for 30 min with IMD receptor antagonist
   IMD[17–47] (10^−6 mol/L; Phoenix Pharmaceuticals), PI3K inhibitor
   LY294002 (10 μmol/L; Sigma, St. Louis, MO, USA), and AMPK inhibitor
   Compound C (10 μmol/L; Sigma) [[314]51] prior to IMD and PA treatment.

4.7. H&E, Sirius Red, and Oil Red O Staining

   The hearts were fixed for 10–12 h, embedded in paraffin and sectioned
   (5 μm), and stained with H&E, as previously described [[315]34]. Other
   sections were stained with a 0.1% Sirius-red picrate solution for 60
   min. The frozen slides were covered with Oil Red O working solution for
   30 min. Images were acquired using a microscope. The stained
   ventricular area was analyzed with ImageJ 1.53k software to quantify
   cardiac fibrosis and the cardiac myocyte cross-sectional area
   [[316]34].

4.8. Immunofluorescence Staining of Cardiac Fibroblast

   Cardiac fibroblasts were fixed for 10 min and washed with PBS. The
   cells were incubated with antibodies for α-SMA (1:100) and Col1a1
   (1:100) at 4 °C overnight. Then, cells were incubated with
   fluorescein-labeled secondary antibody (Millbrae, CA, USA). Nuclei were
   counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen,
   Carlsbad, CA, USA). Immunofluorescent images were acquired using a
   Leica Imaging Systems microscope (Cambridge, UK).

4.9. Detection of Fasting Serum Glucose and Insulin and Homeostasis Model
Assessment for Insulin Resistance (HOMA-IR) Analysis

   Mouse and rat serum were obtained from blood samples, which were
   precipitated under natural static conditions. FBG levels of rats and
   mice were determined with a detection kit (100000240, Zhong Sheng
   Biotechnology, Beijing, China). The enzyme-linked immunosorbent assay
   (ELISA) kit, in accordance with the manufacturer’s instructions
   (E-EL-R3034, E-EL-M1382c, Elabscience, Wuhan, China), were used to
   determine the serum insulin levels of the rats and mice. The HOMA-IR
   was calculated as follows, HOMA-IR = [Insulin (μIU/mL) × Blood glucose
   (mmol/L)]/22.5 [[317]81].

4.10. Quantitation of Serum Lipids in Mice and Rats

   Serum from the rats and mice was obtained from blood samples, which
   were precipitated under natural static conditions at the end of the
   experiment. Total cholesterol (100000180), triglycerides (100000220),
   low-density lipoprotein cholesterol (LDL-C) (100020245), and
   high-density lipoprotein cholesterol (HDL-C) (100020235) detection kits
   were from Zhong Sheng Biotechnology (Beijing, China) and were used to
   determine lipid profiles. The serum of the rats and mice were mixed
   separately with working fluid, and OD values were detected by a
   biochemical analyzer to determine the levels of total cholesterol,
   triglycerides, LDL-C, and HDL-C.

4.11. ELISA

   Rat and mouse serums were obtained from blood samples, which were
   precipitated in natural static conditions at the end of the experiment.
   Cardiomyocytes were stimulated with PA after incubation with IMD. The
   ELISA kits for mouse, rat, and human CPT-1β (MM-46720M2, MM-71573R2,
   MM-61760H1) were purchased from Jiangsu Meimian (Yancheng, China). The
   ELISA kits for mouse, rat, and human acetyl-CoA (ER11934, ER07786,
   ER01745) were purchased from Zancheng (Tianjin, China). We compared the
   O.D. to the standard curve from the manufacturer’s instructions to
   determine CPT-1β enzymatic activity and acetyl-CoA content in rat and
   mouse serum, human plasma, and supernatant of the cardiomyocytes.

4.12. Western Blot Analysis

   Equal amounts of total protein from heart tissues or cell extracts were
   resolved by 10% or 12% SDS-PAGE. We incubated the blots with primary
   antibodies β-actin (sc-47778, Santa Cruz, CA, USA, 1:2000), CRLR
   (GTX64616, GeneTex, Irvine, CA, USA, 1:500), RAMP1 (ab156575, Abcam,
   Cambridge, UK, 1:1000), RAMP2 (sc-365240, 1:300), RAMP3 (sc-365313,
   1:300), Col1a1 (ab260043, 1:500), Col3a1 (22734-1-AP, Proteintech,
   Chicago, IL, USA, 1:500), CPT-1β (A6796, ABclonal, Wuhan, China,
   1:1000), p-AMPK (Thr172) (2531, Cell Signaling Technology, Danvers, MA,
   USA, 1:1000), AMPK (2532, Cell Signaling Technology, 1:1000), p-Akt
   (Ser473) (4060, Cell Signaling Technology, 1:500), Akt (9272, Cell
   Signaling Technology, 1:1000), p-PKA (Thr197) (sc-32968, 1:1000), and
   PKA (sc-32968, 1:1000) overnight, then with secondary antibodies for 1
   h. Protein levels were analyzed with NIH ImageJ 1.53k and normalized to
   β-actin.

4.13. Quantitative Real-Time PCR Analysis

   Total RNA from cardiac tissue or cultured cardiomyocytes was extracted
   with Trizol. The RNA-to-cDNA kit (Novoprotein, Shanghai, China) was
   used to convert RNA to cDNA. The Applied Biosystems 7500 fast PCR
   System (Life Technologies, Carlsbad, CA, USA) and SYBR Green I reagent
   (Novoprotein, Shanghai, China) were used for quantitative real-time PCR
   amplification. Primer sequences for real-time PCR is shown in
   [318]Table S8. The cycle threshold (Ct) reflected the number of PCR
   cycles required for the fluorescent signal to cross a set threshold.
   The 2^−ΔΔCt method was used for relative quantification, with Gapdh as
   a reference.

4.14. RNA Sequencing

   RNA extracted from the hearts of three diabetic WT, IMDtg and IMD^−/−
   mice were used for RNA sequencing. Fresh heart tissues were obtained
   right after killing the mice. We then quickly placed the heart tissues
   in liquid nitrogen. The qualified total RNA was extracted, purified,
   and reverse-transcribed after fragmentation, and the cDNA library was
   constructed. RNA samples (1 μg) were used for library preparation using
   the NEBNext Ultra RNA sample preparation kit (Illumina). Differential
   gene expression analysis was estimated in the DESeq2 R package
   (1.16.1). Genes with an adjusted p value < 0.05 were assigned as
   differentially expressed.

4.15. siRNA Transfection and Identification

   CPT-1β siRNAs (siCPT-1β) and negative scramble siRNA were from RiboBio
   (Guangzhou, China). Using the Lipofectamine RNAiMAX Transfection
   Reagent (Invitrogen, Carlsbad, CA, USA), we incubated NRCMs at 50%
   confluence with CPT-1β siRNA or negative control siRNA at a final
   concentration of 50 nmol for 6 h. After transfecting with serum-free
   Dulbecco’s modified Eagle medium for 6 h, we changed the medium to a
   serum-containing medium. Quantitative RT-PCR and Western blot analysis
   were used to evaluate gene knockdown efficiency after transfection at
   36 h and 72 h, respectively.

4.16. Statistical Analysis

   All the analyses were performed using GraphPad Prism version 7.0
   (GraphPad Software Inc., San Diego, CA, USA). Normal distribution was
   tested using the Shapiro–Wilk test. Homogeneity of variances was
   examined using the Brown–Forsythe test. All data were expressed as the
   mean  ±  SD. Student’s t-test was used to compare two groups, and
   one-way ANOVA or two-way ANOVA, followed by Tukey’s post hoc test, were
   used for multiple group comparisons. A p-value of less than 0.05
   indicated a statistically significant difference.

5. Conclusions

   In conclusion, our study revealed the protective role of the
   cardiovascular bioactive peptide IMD in DCM. IMD may attenuate DCM by
   up-regulating CPT-1β via CRLR/RAMP receptor complexes and PI3K/Akt
   signaling. Targeting IMD as a therapeutic strategy could have potential
   for DCM prevention and treatment.

Supplementary Materials

   The following supporting information can be downloaded at:
   [319]https://www.mdpi.com/article/10.3390/ph17091204/s1, Figure S1. IMD
   and IMD receptor levels are decreased in the hearts of diabetic rats.
   Figure S2. IMD and IMD receptor levels are decreased in the hearts of
   diabetic mice. Figure S3. Verify the levels of IMD in the hearts of
   IMDtg mice and IMD^−/− mice. Figure S4. Expression of CPT-1β after
   administration of CPT-1β siRNA in NRCMs. Figure S5. The identification
   of NRCMs and cardiac fibroblasts. Figure S6. Effects of different
   concentrations and different treatment times of palmitic acid on the
   viability of NRCMs and cardiac fibroblasts. Table S1. Glucose and lipid
   metabolic data of diabetic rats. Table S2. Echocardiographic parameters
   of diabetic WT and IMDtg mice. Table S3. Blood pressure in diabetic WT
   and IMDtg mice. Table S4. Glucose and lipid metabolic data of diabetic
   WT and IMDtg mice. Table S5. Echocardiographic parameters of diabetic
   WT and IMD^−/− mice. Table S6. Blood pressure in diabetic WT and
   IMD^−/− mice. Table S7. Glucose and lipid metabolic data of diabetic WT
   and IMD^−/− mice. Table S8. Primer sequences for real-time PCR.
   [320]pharmaceuticals-17-01204-s001.zip^ (3.5MB, zip)

Author Contributions

   J.Z. and Y.-F.Q. designed and conceived the experiments. J.Z., Y.-R.Z.,
   S.-M.L. and D.-R.J. conducted and analyzed the experiments. J.Z. and
   Y.-F.Q. wrote the manuscript. J.Z., L.H., R.W., Y.-R.Y., M.-Z.J.,
   S.-B.C., H.-F.T., W.H. and Y.-F.Q. participated in the reviewing and
   editing process. All authors have read and agreed to the published
   version of the manuscript.

Institutional Review Board Statement

   Animal studies were conducted according to the guidelines of the Guide
   for the Care and Use of Laboratory Animals published by the US National
   Institutes of Health (NIH Publication, 8th Edition, 2011) and approved
   by the Animal Care Committee of Peking University Health Science Center
   (No. LA2020042).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data presented in this study are contained within the article.

Conflicts of Interest

   The authors declare that they have no conflicts of interest.

Funding Statement

   This work was supported by the National Natural Science Foundation of
   China (32271153, 32071113, and 82370454) and the Beijing Natural
   Science Foundation of China (7212059, 7232226).

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References