Endothelial restoration of receptor activity-modifying protein 2 is sufficient to rescue lethality, but survivors develop dilated cardiomyopathy

Abstract

Receptor activity-modifying proteins (RAMPs) serve as oligomeric modulators for numerous G protein-coupled receptors (GPCRs), yet elucidating the physiological relevance of these interactions remains complex. Receptor activity-modifying protein 2 (Ramp2) null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor (CLR) signaling. We aimed to genetically rescue the Ramp2^−/− lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with Family B GPCRs in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2^−/− mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2^−/− Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking CLR. Yet, the hearts of Ramp2^−/− Tg animals displayed dysregulation of Family B GPCRs, including parathyroid hormone and glucagon receptors as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional GPCR pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/CLR signaling, in which future studies could elucidate novel and pharmacologically-tractable pathways for treating cardiovascular diseases.

Introduction

Receptor activity-modifying proteins (RAMPs) are single-pass transmembrane proteins that physically interact with numerous G protein-coupled receptors (GPCRs) to regulate receptor trafficking, ligand binding specificity and downstream G protein coupling and signaling. Biochemical and pharmacological studies have revealed functional RAMP interactions with many GPCRs including, calcitonin receptor-like receptor (CLR = protein, Calcrl = gene), calcitonin receptor (CTR), parathyroid hormone receptor 1 and 2 (PTHR1 and PTHR2), glucagon receptor (GCGR), secretin receptor (SCTR), vasointestinal peptide receptor 1 and 2 (VIPR1 and VIPR2), calcium sensing receptor (CaSR), an estrogen receptor (GPR30), and likely additional RAMP-interacting GPCRs will be identified in the future.^1–4 Thus, in their capacity as endogenous oligomeric GPCR partners, RAMPs can exert powerful modulation on nearly every aspect of GPCR function and on a wide range of physiological processes. However, our understanding regarding the complexity and consequences of RAMP-GPCR interactions in vivo lags behind our current structural, biochemical and pharmacological knowledge and thus presents a limitation toward harnessing the potential pharmacological utility of this family of proteins.

There exist three mammalian RAMPs, each encoded by single genes, which have been systematically knocked out by our laboratory and others.^5–7 As expected, based on their broad tissue distribution, the phenotypes of Ramp null mice are extensive and largely reflect their interactions with multiple GPCRs.⁸ Ramp1 and Ramp3 null mice survive to adulthood, while Ramp2^−/− mice exhibit embryonic lethality at mid-gestation due to cardiovascular defects in the heart, blood, and lymphatic vasculatures.^{5–7, 9–11} These phenotypes are essentially identical to those observed in mice lacking the genes that encode for CLR and its Ramp2-associated ligand, adrenomedullin (AM), indicating that RAMP2 is essential for canonical CLR/AM signaling during embryonic development. Yet, mice that are haploinsufficient for Ramp2 also exhibit an expanded constellation of endocrine-related phenotypes that are not observed in CLR and AM haploinsufficient mouse models.¹² Therefore, these data imply that RAMP2 must exert functional modulation of other GPCRs, which is supported by its in vitro biochemical interaction with CTR, PTHR1/2, GCGR, and VIPR1/2.² To date, ascribing a functional relevance for these other putative RAMP2-interacting GPCR pathways in adult physiological systems has been precluded by the embryonic lethality of Ramp2^−/− mice.

Here, we have developed a novel Ramp2 transgenic mouse model which overexpresses Ramp2 specifically in the endothelium, with the hypothesis that restoration of RAMP2 in the endothelium will rescue the lethality-causing vascular defects associated with loss of CLR/AM signaling during embryogenesis. Indeed, endothelial RAMP2 is sufficient to rescue the embryonic lethality of many Ramp2^−/− mice. Thus, the surviving mice, which express Ramp2 in the endothelium but lack Ramp2 in every other cell type, provided us with the opportunity to further elucidate the functional implications of the loss of Ramp2 on adult cardiovascular physiology and respective changes in other Ramp2-associated GPCRs.

Methods

Mouse Studies

Previously published SvEv-Ramp2^+/− and Calcrl^flox/flox mice were both fully backcrossed (>10 generations) onto a C57BL/6J genetic background for these studies.^{5, 9} A novel endothelial-specific Ramp2 transgenic mouse was generated and crossed to the Ramp2^+/− mice. All Ramp2 mice used in this study were maintained on an isogenic C57BL/6J genetic background. The Calcrl^flox/flox mice were crossed with either the SJL-Tg(Tagln-cre)1Her (SM22-Cre) or the inducible CAGG-CreER^TM mouse to conditionally delete Calcrl specifically within developmental vascular smooth muscle cells (vSMC) or ubiquitously in adult mice, respectively. Adult CAGG-CreER^TM females were administered with tamoxifen (TAM) as previously published.¹³ Genotyping and RT-PCR primers and probes are listed in Supplemental Table S1. Females between 4–8 months of age were used in all Ramp2-associated studies, males 3–4 months of age for vSMC-specific Calcrl studies, and females 16 month old for ubiquitous Calcrl studies. Mice were acclimated and conscious for both non-invasive tail cuff blood pressure and echocardiography analysis and anesthetized with isoflurane for intra-arterial blood pressure measurements. Organs weights and heart chamber dissections were normalized to either body weight or tibia length as previously described.¹⁴ Biological N of 3–10 mice per genotype was used for each experiment. Endothelial cells from adult mice were isolated with magnetic-associated cell sorting using CD31-specific antibodies as previously described.¹⁵ Cardiomyocytes from adult mice were isolated using collagenase digestion as previously described.¹⁶ All animal experiments were approved by the Institutional Animal Care and Use Committee of The University of North Carolina at Chapel Hill.

Statistical Analysis

Statistical analysis was determined with GraphPad 5.0 and data is represented as mean ± SEM. The unpaired student T-test was used to compare two groups, while one way ANOVA with Tukey’s Multiple Comparison test was used to compare 3 groups. Survival data were compared by Mantel-Cox and Gehan-Breslow-Wilcoxon tests. Significant differences are represented as * p <0.05, ** p <0.01, ***p <0.001.

Additional detailed methods are provided in the Online Supplemental Data.

Results

Generation and Characterization of Endothelial-Specific Ramp2 Transgenic Animals

A diagram of the Cdh5-Ramp2 transgene (Tg) depicts the murine vascular cadherin 5 (Cdh5) promoter driving the expression of a flagged-tagged murine Ramp2 cDNA (Supplemental Figure S1A), which was successfully integrated into the genome of two independent founder lines on the C57BL/6J genetic background. Expression of FLAG-Ramp2 protein was confirmed using both transgenic founder lines in a variety of adult tissues including the heart, kidney, lung and intestine (Supplemental Figure S1B). Semi-quantitative RT-PCR revealed the presence of FLAG transcript within whole adult lung tissue and CD31+ endothelial-enriched cells in the Tg animals, but not wildtype endothelium (Supplemental Figure S1C), which resulted in a modest increase in overall Ramp2 gene expression levels within lung CD31+ endothelial cells of Tg animals compared to wildtype animals (Supplemental Figure S1D). Ramp2 expression was significantly higher in the left ventricles of Tg animals compared to wild-type—a finding which we attribute to the endothelial-driven transgene expression, as compared to the low levels of endogenous Ramp2 expression in isolated cardiomyocytes (Supplemental Figure S1E) and previously reported expression in vSMCs.^{17, 18} Tg(Cdh5-Ramp2) mice bred normally, with expected Mendelian and sex ratios at birth, and no obvious phenotypic defects.

Transgenic Endothelial Ramp2 Partially Rescues Embryonic Lethality of Ramp2^−/− Mice

We sought to determine whether endothelial Ramp2 expression could rescue the previously reported Ramp2^−/− embryonic lethality by interbreeding the hemizygous Tg(Cdh5-Ramp2) animals with Ramp2^+/− non-transgenic (ntg) animals, on an isogenic C57BL/6J genetic background.^{7, 9} Ramp2^+/− Tg(Cdh5-Ramp2) mice (hereafter referred to as Ramp2^+/− Tg) were crossed to Ramp2^+/− ntg animals to generate Ramp2^−/− embryos with or without expression of the transgene. At e14.5, all six possible genotypes were observed (Supplemental Figure S2A). As expected, embryos lacking Ramp2 display non-hemorrhagic edema associated with arrested lymphangiogenesis,⁹ yet the Ramp2^−/− Tg embryos showed significantly less edema than Ramp2^−/− ntg littermates (Figure 1A–B). Unlike the small, hypoplastic jugular lymph sacs of Ramp2^−/− ntg embryos, the jugular lymph sacs of the Ramp2^−/− Tg were comparable in size to those of Ramp2^+/+ littermates (Figure 1C–D), demonstrating that transgenic restoration of functional Ramp2 in the endothelium leads to improvement of the edematous phenotype.

Figure 1. Endothelial restoration of Ramp2 partially rescues embryonic edema leading to prolonged survival of Ramp2^−/− mice.

(A) Representative images and (B) quantification of edema severity from e14.5 Ramp2^+/+, Ramp2^−/− ntg, and Ramp2^−/− Tg embryos. Edema scoring system: (1) no edema, (2) mild edema, and (3) severe edema. (C) Immunohistochemistry and (D) quantification of jugular lymph sac size using Lyve1 (green) and DAPI (magenta). CA, carotid artery; JV, jugular vein; JLS, jugular lymph sac. (E) Histology of descending aortas and (F) quantification of aortic vSMC wall thickness. (G) Heart histology, (H) quantification of ventricle area, and (I) whole heart relative Ramp2 expression from viable Ramp2^+/+ ntg, Ramp2^−/− ntg, and Ramp^−/− Tg e14.5 embryos. Samples were normalized to Ramp2^+/+ ntg and Gapdh expression. Scale bars: 100 μm. Data represented as average ± SEM from N = 3–8 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison with *p<0.05, **p<0.01, and ***p<0.001.

In addition to defects in lymphatic development, loss of Ramp2 or AM/CLR signaling leads to small, disorganized hearts and thin vSMC walls.^{9, 19, 20} The aortic endothelium of viable embryos appeared intact with no signs of hemorrhagic leakage or endothelial dysfunction as previously reported in some Ramp2^−/− embryos.⁷ However, the aortic vSMC layer was significantly thinner in Ramp2^−/− ntg and Ramp2^−/− Tg embryos (Figure 1E–F). Likewise, the hearts of e14.5 Ramp2^−/− Tg animals were comparable in size to Ramp2^−/− ntg littermates, which were both significantly smaller than controls, despite detectable expression of Ramp2 in Ramp2^−/− Tg hearts (Figure 1G–I). Consistent with previous reports, these data reflect that Ramp2 expression is required for normal vSMC and cardiac development and that endothelial Ramp2 restoration is not sufficient to rescue these defects, thus confirming non-endothelial roles of Ramp2 during cardiovascular development.

Unlike the Ramp2^−/− ntg embryos, which uniformly die by e15.5, Ramp2^−/− Tg embryos survive to term at near-Mendelian ratios, but a large number of these animals were found stillborn at postnatal day 1 (Supplemental Figure S2B). Nevertheless, by postnatal day 7, 40% of the expected Mendelian ratio of Ramp2^−/− Tg pups were viable, which represents a significant survival rescue when compared to the completely penetrant lethality of Ramp2^−/− ntg mice (Table 1). These surviving Ramp2^−/− Tg mice were indistinguishable from Ramp2^+/+ littermates (Supplemental Figure S2C). Interestingly, there was a significant skewing in the sex ratio of the surviving Ramp2^−/− Tg mice, such that 78% were female. These data demonstrate that transgenic endothelial restoration of Ramp2 is able to blunt the endothelial and edematous phenotypes observed with global Ramp2 genetic deletion, leading to significantly improved survival.

Table 1. Endothelial restoration of Ramp2 can rescue the global Ramp2^−/− embryonic lethality.

Breeding results, both expected Mendelian and actual results from Ramp2^+/− Tg(Cdh5-Ramp2) crossed with Ramp2^+/− ntg. N = 280 pups.

Mendelian Ratio	Ramp2+/+	ntg Ramp2+/−	Ramp2−/−	Tg(Cdh5-Ramp2)
				Ramp2+/+	Ramp2+/−	Ramp2−/−
Expected N	35	70	35	35	70	35
Actual N P7	47	88	0	48	83	14*
Actual/Expected	134%	126%	0%	137%	119%	40%

Significance in survival between Ramp2^−/− ntg and Ramp2^−/− Tg pups determined by the Mantel-Cox test:

^*p<0.001.

Surviving Ramp2^−/− Tg Adult Mice are Hypotensive

The surviving Ramp2^−/− Tg mice, which express Ramp2 in the endothelium but lack Ramp2 in all other cells, provided an opportunity to evaluate how Ramp2 loss-of-function during development affects adult cardiovascular homeostasis. Diastolic, systolic, and mean arterial blood pressures were significantly reduced in Ramp2^−/− Tg compared to Ramp2^+/+ ntg and Tg female controls (Figure 2A). Importantly, there were no significant differences in basal blood pressure between Ramp2^+/+ ntg and Ramp2^+/+ Tg mice, indicating that the decreased blood pressure was not caused solely by the transgene. Moreover, the reduced basal blood pressure of Ramp2^−/− Tg mice was observed without the exogenous administration of the canonical, hypotensive ligand AM, which was previously required to lower blood pressures in a vSMC-specific overexpression model of Ramp2.^{21, 22}

Figure 2. Ramp2^−/− Tg adults are hypotensive.

(A) Tail-cuff telemetry blood pressure analysis from conscious Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg adult female mice. (B) Representative images and vSMC wall thickness quantifications of Ramp2^+/+ ntg and Ramp2^−/− Tg descending aortas. Scale Bars: 100 μm. (C) Intra-arterial blood pressure measurements from anesthetized Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg mice during baseline and after challenged with intravenous bolus of 30 μg/kg phenylephrine (PE) or 12 nmol/kg adrenomedullin (AM). Drug response represented as a % change from baseline (dotted line). Data represented as average ± SEM from N = 3–5 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison (A, C) or unpaired student T-test (B) with *p<0.05 and **p<0.01.

Similar to the developmental defects in vSMC wall thickness of the Ramp2^−/− Tg embryos and other models lacking AM/CLR/Ramp2 function,^{9, 19, 20} the vSMC walls were significantly thinner in Ramp2^−/− Tg adult descending aortas compared to wild-type controls (Figure 2B). Intra-arterial blood pressure measurements further confirmed the hypotensive phenotype of adult Ramp2^−/− Tg females (Figure 2C). To test vSMC responsiveness, Ramp2^−/− Tg and controls were challenged with intravenous injections of the α-adrenergic receptor agonist, phenylephrine, and the vasodilator, AM. The Ramp2^−/− Tg adults were capable of normal vasoconstriction and vasodilation as compared to controls (Figure 2C). So, although the thinner vSMC walls likely play a role in the hypotensive phenotype of Ramp2^−/− Tg mice, additional systemic changes likely contribute to the hypotension.

Ramp2^−/− Tg Mice Develop Spontaneous Dilated Cardiomyopathy Phenotype

Considering the hypotension and the developmental defects observed in Ramp2^−/− Tg embryonic hearts, we next assessed how cardiac function and morphology were altered in adult Ramp2^−/− Tg mice. Echocardiography on conscious mice revealed significantly dilated left ventricles during both diastole and systole, with significantly larger left ventricle volumes and left ventricle internal diameter dimensions in the Ramp2^−/− Tg mice compared to Ramp2^+/+ and Ramp2^+/−, with and without the transgene (Table 2). Representative M-mode echocardiograms from Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg illustrate the ventricular dilation in Ramp2^−/− Tg mice (Figure 3A), which resulted in a trending increase in calculated cardiac output in these animals (Table 2). Septum and left ventricle posterior wall dimensions of Ramp2^−/− Tg mice were unchanged from controls, although there was a non-significant trend towards a thinner posterior wall. There was also a modest, but significant reduction in both ejection fraction and fractional shortening in the Ramp2^−/− Tg dilated hearts. These data indicate that loss of Ramp2 in non-endothelial cells leads to a spontaneous dilated cardiomyopathy (DCM)–like phenotype in adult mice, which at 6 months of age had not yet progressed to heart failure, as indicated by the elevated cardiac output and sufficient heart function.

Table 2. Ramp2^−/− Tg adults develop left ventricle dilatation and declining heart function.

Echocardiography analysis from Ramp2^+/+, Ramp2^+/−, and Ramp2^−/− mice with and without the transgene.

Echo Parameters	ntg		Tg (Cdh5-Ramp2)
	Ramp2+/+	Ramp2+/−	Ramp2+/+	Ramp2+/−	Ramp2−/−
Mouse Number	10	4	7	4	7
Mouse Age (wks)	26.7±1.3	26.8±2.0	29.3±2.3	26.3±2.2	29.7±1.6
Heart Rate (BPM)	624±15	578±38	574±50	614±55	552±21
LV Vol, d (μL)	35.6±4.7	32.0±3.9	34.1±6.7	27.9± 5.9	73.4±9.5*,†,‡,§
LV Vol, s μL)	7.6±1.6	5.0±1.4	7.0±2.7	5.4±2.4	23.1±3.5*,†,‡,§
CO (mL/min)	16.5±2.1	15.4±1.2	16.1±3.2	13.2±0.9	27.0±4.8*
EF (%)	81.2±3.0	85.1±2.0	82.4±3.6	83.3±5.1	68.3±2.3*,†,‡,§
FS (%)	51.6±2.7	52.9±2.2	51.1±4.0	52.4±6.5	37.9±1.8*,‡
IVS, s (mm)	1.13±0.05	1.16±0.06	1.12±0.07	1.07±0.07	1.08±0.07
IVS, s (mm)	1.72±0.06	1.74±0.06	1.73±0.07	1.68±0.13	1.60±0.07
LVID, d (mm)	2.94±0.20	2.88±0.14	2.90±0.22	2.70±0.23	4.03±0.22*,†,‡,§
LVID, s (mm)	1.52±0.17	1.36±0.13	1.45±0.21	1.32±0.27	2.50±0.14*,†,‡,§
LVPW, d (mm)	1.54±0.10	0.96±0.07	1.22±0.13	1.08±0.30	0.87±0.06
LVPW, s (mm)	1.74±0.13	1.72±0.04	1.76±0.08	1.60±0.20	1.40±0.08

BPM, beats per minute; LV, left ventricle; d, diastole; s, systole; CO, cardiac output; EF, ejection fraction; FS, fractional shortening; IVS, interventricular septal; LVID, left ventricle internal diameter; LVPW, left ventricle posterior wall. Data represented as averages ± SEM with significance of p<0.05 determined by one-way ANOVA with Tukey’s Multiple Comparison. Significant compared to:

^*Ramp2^+/+ ntg,

^†Ramp2^+/− ntg,

^‡Ramp2^+/+ Tg,

^§Ramp2^+/− Tg.

Figure 3. Ramp2^−/− Tg adults develop spontaneous dilated cardiomyopathy.

(A) Representative M-mode echocardiograms from left ventricles of Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg mice. Macroscopic (B) and H&E (C) images of whole hearts and left ventricles from of Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg mice. Scale Bars: 2 mm. Quantification of heart (D), left ventricle (E), and right ventricle (F) weight normalized to body weight. Data represented as average ± SEM from N = 4–10 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison with *p<0.05, **p<0.01, ***p<0.001.

Upon dissection, we observed that the hearts of Ramp2^−/− Tg mice were grossly enlarged compared to wildtype and Ramp2^−/− ntg mice (Figure 3B–C). The adult Tg mice had no differences in body weight (Ramp2^+/+ ntg: 28.4 ± 1.4 g, Ramp2^+/+ Tg: 28.4 ± 1.2 g, Ramp2^−/− Tg: 28.3 ± 1.2 g) or tibia length (Ramp2^+/+ ntg: 17.6 ± 0.2 mm, Ramp2^+/+ Tg: 17.9 ± 0.1 mm, Ramp2^−/− Tg: 17.8 ± 0.2 mm) regardless of their Ramp2 genotype. Yet, total heart weights were significantly larger in the Ramp2^−/− Tg mice when normalized to both body weight (Figure 3D) or tibia length (Ramp2^+/+ ntg: 7.3 ± 0.4 mg/mm, Ramp2^+/+ Tg: 6.5 ± 0.2 mg/mm, Ramp2^−/− Tg: 9.0 ± 0.3** mg/mm; **p<0.01). Moreover, when normalized to body weight the left ventricle (Figure 3E), right ventricle (Figure 3F), and right atria (Ramp2^+/+ ntg: 0.15 ± 0.01 mg/g, Ramp2^+/+ Tg: 0.15 ± 0.01 mg/g, Ramp2^−/− Tg: 0.21 ± 0.02* mg/g; *p<0.05) all exhibited significant enlargement in the Ramp2^−/− Tg animals compared to all other genotypes. Similar significant trends were observed when the individual chamber weights were normalized to tibia length (LV:TL; Ramp2^+/+ ntg: 5.4 ± 0.3 mg/mm, Ramp2^+/+ Tg: 4.8 ± 0.2 mg/mm, Ramp2^−/− Tg: 6.5 ± 0.2** mg/mm; **p<0.01).

Cross-sectional area of myocytes within the left ventricle revealed slight, but significant cardiomyocyte hypertrophy, with no changes in left ventricular capillary density, in the Ramp2^−/− Tg mice compared to controls (Figure 4A–D). Picrosirius red staining showed no differences in perivascular fibrosis, but there was significantly increased interstitial fibrosis in the Ramp2^−/− Tg hearts compared to hearts of Ramp2^+/+ and Ramp2^+/+ Tg mice (Figure 4E–H). There were elevated levels of the oxidative stress indicator, lipid peroxidase evidenced by 4-hydroxynonenal (4-HNE) staining in Ramp2^−/− Tg hearts (Figure 4I–J).²³ Together, the modest increases in hypertrophy, fibrosis, and oxidative stress, as well as modest decline in heart function, further support that 6 month old Ramp2^−/− Tg mice exhibit a compensated, DCM-like phenotype.

Figure 4. Ramp2^−/− Tg left ventricles hypertrophied with early fibrotic and oxidative stress changes.

(A) Histology and (B) wheat germ agglutinin (WGA, white) immunohistochemistry showing cardiomyocytes from Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg left ventricles. Quantification of (C) cardiomyocyte cross-sectional area and (D) capillary density from mouse left ventricles. Representative images and quantification of left ventricles stained with Picrosirius Red showing (E–F) perivascular and (G–H) interstitial fibrosis/collagen deposition. (I) Immunohistochemistry and (J) quantification of 4-HNE (white) staining showing relative levels oxidative stress in left ventricles. Scale Bars: 100 μm. Data represented as averages ± SEM from N = 3–4 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison with *p<0.05 and **p<0.01.

Adult Ramp2^−/− Tg Mice Develop Multi-Organ Inflammation

DCM and hypotension can lead to vascular congestion and organ dysfunction throughout the body.^24–26 Consistently, we observed that the spleen to body weight ratio was significantly increased in the Ramp2^−/− Tg mice compared to all other genotypes (Figure 5A). Ramp2^−/− Tg mice also exhibited macroscopic vascular congestion within their livers compared to control animals (Figure 5B). Furthermore, multi-organ histology revealed a marked increase in the number of inflammatory foci, particularly surrounding the vasculature, within the liver (Figure 5C–D), kidney (Figure 5E–F) and lungs (Figure 5G–H). A diagnostic profile of adult serum from wildtype and Ramp2^−/− Tg mice revealed few significant changes in circulating ions or enzyme levels that are typically indicative of hepatocellular or renal damage (Supplemental Table S2). This supports that the end-organ inflammation in Ramp2^−/− Tg mice is likely downstream of altered hemodynamics due to DCM and hypotension, rather than primarily due to loss-of-function of Ramp2 in end-organs.

Figure 5. Ramp2^−/− Tg have vascular congestion and multi-organ inflammation downstream of hypotension and dilated cardiomyopathy.

(A) Spleen to body weight ratio from Ramp2^+/+, Ramp2^+/−, and Ramp2^−/− females with and without the transgene. (B) Macroscopic images of liver vasculature abnormalities in Ramp2^−/− Tg compared to that of Ramp2^+/+ ntg and Ramp2^+/+ Tg controls. Histology and quantification of inflammatory foci (blue arrows) in (C–D) livers, (E–F) kidneys, and (G–H) lungs from Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg mice. Scale Bars: 1 mm (B) and 100 μm (C, E, G). Data represented as averages ± SEM from N = 3–4 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison with *p<0.05, **p<0.01, ***p<0.001.

Conditional Calcrl Deletion Does Not Lead to a Dilated Cardiomyopathy Phenotype

Since CLR and AM represent the most well-characterized, canonical pathway for RAMP2 modulation and the knockout mice for these genes recapitulate the Ramp2^−/− developmental phenotypes, it is reasonable to consider that disruption of this pathway, which has been demonstrated to be cardioprotective in both animal studies and in humans,^27–30 may underlie the DCM and hypotensive phenotypes in Ramp2^−/− Tg mice. To test the functions of CLR in non-endothelial cells, we generated cardiac- and vSMC-specific Calcrl null mice using the SM22-Cre mediated excision. The Calcrl^loxP/loxP; SM22Cre+ mice were born at expected Mendelian ratios and survived to adulthood (Supplemental Figure S3A). Relative Calcrl expression was significantly reduced in aortic vSMCs and hearts of Calcrl^loxP/loxP; SM22-Cre+ compared to Calcrl^loxP/+; SM22-Cre+ littermates (Supplemental Figure S3B). Importantly, Calcrl^loxP/loxP; SM22-Cre+ adult males were normotensive and had no basal changes in heart function, size, or morphology (Supplemental Table S3 or Figure S3C). Similarly, cardiac-specific Calcrl deletion using the αMHC-Cre+ transgenic line also failed to recapitulate the DCM phenotype, with animals surviving to adulthood with no basal cardiac dysfunction (Dackor R & Caron K.M., unpublished data, [2016]). These data demonstrate that cardiac- and vSMC-specific Calcrl expression is not required for embryonic development or adult cardiovascular maintenance.

We have previously shown that temporal, global deletion of Calcrl in adult Calcrl^loxP/loxP; CAGG-CreER^TM mice results in dilated lymphangiectasia in many lymphatic vascular beds throughout the body.¹³ However, these mice did not display any obvious cardiac phenotypes and they had similar heart to body weight ratio as TAM-injected control mice (Calcrl^loxP/loxP; CAGG-CreER^TM: 4.43 ± 0.19 mg/g versus Calcrl^flox/flox: 4.13 ± 0.14 mg/g, respectively). Furthermore, conscious echocardiography of Calcrl^loxP/loxP; CAGG-CreER^TM female mice, even at 14 months of age, failed to reveal any significant changes in left ventricle internal diameter, function, or heart size compared to control mice (Supplemental Table S4). Collectively, these data, generated from three independent series of conditional deletion approaches, indicate that the global-, cardiac- or vSMC-specific loss of Calcrl does not recapitulate the DCM phenotype observed in the Ramp2^−/− Tg animals. Therefore, this strongly suggests that the Ramp2^−/− Tg phenotype is imparted by other RAMP2-associated GPCR pathways.

Ramp2^−/− Tg hearts exhibit reduced signaling pathways and expression of numerous RAMP-associated GPCRs

The genetic reduction or down-regulation of the transcriptional regulator CREB^{25, 31} and the crucial PPAR pathway transcription factor, Pgc-1α,^32–34 have both been shown to be involved in DCM pathogenesis. Thus as expected, the relative expression of Pgc-1α was significantly down-regulated in left ventricles and in an enriched cardiomyocytes fraction of Ramp2^−/− Tg hearts compared to controls (Figure 6A–B). There was also a significant reduction in phosphorylated CREB compared to total CREB and Gapdh in the Ramp2^−/− Tg hearts compared to Ramp2^+/+ ntg and Ramp2^+/+ Tg hearts (Figure 6C). Numerous Family B GPCRs signal through these pathways, including the Gcgr which has been shown to signal through cAMP to activate both CREB and PPAR transcription,^35–38 and both the Pthr1 and CaSR signal through cAMP and CREB.^39–41 Interestingly, the gene expression levels of these RAMP-associated GPCRs were significantly down-regulated in the left ventricles of Ramp2^−/− Tg mice compared to those of control animals, whereas genes encoding for other RAMP-associated GPCRs, like Calcr, Vipr1, and Gpr30, were unchanged (Figure 6D).

Figure 6. Decreased signaling and expression of RAMP-associated GPCRs in embryonic and adult Ramp2^−/− Tg hearts.

RT-PCR showing relative Pgc-1α expression from Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg adult left ventricle (A) or cardiomyocytes (B). (C) Quantification and representative western blot of phosphorylated CREB to total CREB in Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg left ventricles. Samples were normalized to Ramp2^+/+ ntg and CREB with Gapdh used as a loading control. Relative expression of the Family B GPCRs; Calcrl, Calcr, Pthr1, Gcgr, Vipr1, CaSR and Gpr30 in (D) whole left ventricles and (E) isolated cardiomyocytes from adult Ramp2^−/− Tg mice and Ramp2^+/+ controls. (F) Relative GPCR expression from e14.5 Ramp2^+/+ ntg, Ramp2^−/− ntg, and Ramp2^−/− Tg whole hearts. Samples were normalized to Ramp2^+/+ ntg and Gapdh and Rpl19 expression. (E) Data represented as averages ± SEM from N = 3–5 mice per genotype. Significance determined by one-way ANOVA with Tukey’s Multiple Comparison (A, C, D, F) or unpaired student T-test (B, E) with *p<0.05. (F) Model summarizing cardiovascular phenotypes in Cdh5+ endothelium and Cdh5- cells in Ramp2^+/+ ntg, Ramp2^+/+ Tg, and Ramp2^−/− Tg adults.

Similar gene expression changes in these Family B GPCR expression profiles were confirmed in isolated cardiomyocyte fractions (Figure 6E), further supporting the myocyte-specific genetic dysregulation. Calcrl expression was significantly increased in left ventricle, but not in the cardiomyocyte-enriched fraction, demonstrating that Calcrl upregulation is from a non-cardiomyocyte cell type. The expression of both Pthr1 and Gcgr were significantly decreased during development in the hearts of Ramp2^−/− ntg and Ramp2^−/− Tg embryos (Figure 6F), supporting that these changes are specific to Ramp2 loss rather than secondary to the Cdh5-driven Ramp2 Tg or the DCM phenotype, as might be the case for CaSR. Serum analysis revealed no significant dysregulation of circulating calcium or glucose, thereby eliminating uncompensated Pthr1 or Gcgr systemic signaling as a cause for the cardiovascular phenotypes (Supplemental Table S2). Collectively, these data demonstrate that genetic loss of Ramp2 in non-endothelial cells of the heart leads to down-regulation of both RAMP2-associated GPCRs, Gcgr and Pthr1, as an underlying mechanistic basis for the decreased pCREB and Pgc-1α responsible for the pathogenesis of the DCM-like phenotype in Ramp2^−/− Tg mice (Figure 6G).

Discussion

In this study, we generated an endothelial-specific Ramp2 Tg mouse model to attempt to rescue the embryonic lethality due to global loss of Ramp2. While no Ramp2 null mice survive to birth, we observed a significant number of Ramp2^−/− Tg born and able to survive into adulthood. This result further confirms that endothelial Ramp2 is essential for embryonic survival and represents, to our knowledge, the first genetic rescue of the global Ramp2 null lethality.

It remains unclear why a significant number of Ramp2^−/− Tg pups survive to late-gestation, but are stillborn. Interestingly, it was recently shown that mice with endothelial excision of Ramp2 during development using a Cdh5-Cre died during late-gestation.²⁴ Additionally, they report that approximately 5% of the endothelial Ramp2 knockouts live into adulthood and develop large hearts, hypotension, and multi-organ vasculitis. In this current study we found that 40% of Ramp2^−/− Tg survived to adulthood, and also developed similar cardiovascular and inflammatory phenotypes. These two mouse models, as well as a previously published endothelial-specific deletion of Calcrl, demonstrate that adequate levels and timing of endothelial Ramp2/CLR/AM signaling are critical for embryonic survival.⁹

In addition, these studies identify potential sex-dependent mechanisms of Ramp2, or Cdh5, regulation and function, as evidenced by the substantially reduced numbers of male Ramp2^−/− Tg survivors. We have previously shown that adult Ramp2^+/− females have endocrine phenotypes not present in Ramp2^+/− males.¹² In addition, Ramp3^−/− males, but not females, displayed exacerbated cardiovascular phenotypes when challenged with hypertension.^{4, 10} Thus, while RAMP2 and RAMP3 interact with both similar and different GPCRs, our observations of sex-dependent phenotypes in these genetic animals will provide an area for interesting future investigations.

The complex compensatory mechanisms through which RAMP-mediated AM/CLR signaling regulate blood pressure have not been fully elucidated. It is well documented that AM infusion acts to lower blood pressure through both vSMC and endothelium, but it remains disputed if altered AM expression using genetic models alters basal blood pressure.^{18, 42–45} Moreover, while developmental loss of Ramp2 leads to spontaneous hypotension and adult Ramp1^−/− mice develop hypertension.^{11, 24} Calcrl deletion in vSMC and cardiomyocytes appears dispensable for regulation of basal vascular tone in adult males. It is evident that Ramp2 and AM signaling plays a role in vSMC and myocardium development and function, which likely contributes to the pathogenesis of hypotension and DCM in Ramp2^−/− Tg survivors. Yet, the hypotension and DCM phenotypes occur despite maintenance of normal cardiac output. It is further possible that the developmentally-induced thin vSMC walls contribute to reduced vascular tone; however we demonstrated their ability to effectively respond to acute phenylephrine vasoconstriction. Therefore, additional studies that explore the entire repertoire of RAMP-mediated GPCRs will be required for full elucidation of the mechanistic basis for the hypotension in Ramp2^−/− Tg survivors.

Loss of Ramp2 in multiple non-endothelial cardiac cells, along with altered humoral signaling could lead to cardiac dysfunction and DCM pathogenesis. The genetic dysregulation in isolated cardiomyocytes suggests roles of Ramp2/GPCRs specifically in cardiomyocytes. Interestingly, a recently published study demonstrated that a cardiomyocyte-specific Ramp2 deletion led to a DCM-like phenotype due to mitochondrial dysfunction and irregular calcium handling, which the authors attribute to loss of CLR/AM signaling.⁴⁶ Similarly, this study suggests that cardiomyocyte loss of Ramp2 in the Ramp2^−/− Tg mice is likely responsible for the DCM. Moreover, our data demonstrates that neither cardiomyocyte- or vSMC-specific loss of Calcrl during development or conditional Calcrl deletion in adults recapitulate the DCM phenotype observed in the Ramp2^−/− Tg and the aforementioned Ramp2^flox/flox; αMHC-MerCreMer mice.⁴⁶ Therefore, collectively, these studies imply the involvement of other RAMP-associated GPCRs.

It is apparent that RAMPs interact with numerous GPCRs biochemically, although in vivo physiologic evidence of these interactions is limited. We found that lack of Ramp2 in non-endothelial cells leads to decreased expression of cardiac Pthr1 and Gcgr. Interestingly, human PTHR1 and GCGR interact specifically with RAMP2 and not RAMP1 or RAMP3 in which RAMP2 is important in chaperoning both GPCRs to the plasma membrane.² Furthermore, it was recently demonstrated that RAMP2 was important in GCGR ligand selectivity between glucagon and glucagon-like peptide 1, which have opposing physiologic effects on glucose homeostasis and cardiovascular function.⁴⁷ While GCGR has not been directly connected to DCM pathogenesis, glucagon can alter calcium signaling in myocytes and glucagon-like peptide 1 improves glucose uptake and survival of canines with DCM.^{35, 48} Likewise PTHR1 signaling is important in vitamin D and calcium homeostasis, which have both been associated with DCM.^{49, 50} Together, Ramp2 loss-of-function can not only alter both Pthr1 and Gcgr expression simultaneously, but may also alter their ability to reach plasma membrane, bind ligands, and signal. Additionally, we observed decreased signaling through CREB and downregulation of Pgc-1α, which both have been shown to lead to DCM.^{25, 31, 32} Both Pthr1 and Gcgr signal through CREB and murine deletion of Gcgr led to decreased phosphorylated CREB and Pgc-1a expression levels.^{36, 38} Thus, loss of Ramp2 likely has numerous mechanisms for the development and maintenance of cardiac functions both dependent and independent of canonical AM/CLR/Ramp2 signaling.

Perspectives

The in vivo interplay between GPCR/RAMP/Ligand is highly complex and is only starting to be understood. However, a better understanding of these interactions in a spatial and temporal manner in a pathophysiologic context will help us better target the unique GPCR/RAMP interfaces to potentially treat disease like DCM and hypertension.

Novelty and Significance.

What Is New?

• Using a novel genetic mouse model, we find that endothelial expression of receptor activity-modifying protein 2 (Ramp2) is able to rescue the embryonic lethality of Ramp2^−/− mice.

• Ramp2 loss-of-function leads to spontaneous hypotension, dilated cardiomyopathy, and multi-organ inflammation, which we show are not recapitulated in genetic models with loss of calcitonin-receptor-like receptor (CLR) signaling.

• In vivo loss of Ramp2 causes dysregulation of numerous RAMP-associated GPCRs, including the glucagon and parathyroid hormone receptors.

What is Relevant?

• The physiological consequences of RAMP interactions with Family B GPCRs have been challenging to address due to the embryonic lethality of RAMP2 mice. Here, we elucidate the importance of RAMP2 interactions with several GPCR pathways, which can ultimately provide novel targets, or predict off-target consequences, for pharmacological therapies against cardiovascular disease.

Summary

This study provides genetic in vivo evidence that endothelial Ramp2 expression is necessary and sufficient to rescue the lethality of global loss of Ramp2. Ramp2 expression in non-endothelial cells during development is required to maintain adult blood pressure and cardiac homeostasis—a process that involves numerous GPCR signaling pathways, including glucagon and parathyroid hormone receptor signaling. Collectively, these studies extend the functional repertoire of RAMP-associated receptors in cardiovascular physiology.