Novel Regulation of Cardiac Metabolism and Homeostasis by the Adrenomedullin-Receptor Activity-Modifying Protein 2 System
Adrenomedullin (AM) was identified as a vasodilating and hypotensive peptide mainly produced by the cardiovascular system. The AM receptor calcitonin receptor-like receptor associates with receptor activity-modifying protein (RAMP), one of the subtypes of regulatory proteins. Among knockout mice (−/−) of RAMPs, only RAMP2−/− is embryonically lethal with cardiovascular abnormalities that are the same as AM−/−. This suggests that the AM-RAMP2 system is particularly important for the cardiovascular system. Although AM and RAMP2 are highly expressed in the heart from embryo to adulthood, their analysis has been limited by the embryonic lethality of AM−/− and RAMP2−/−. For this study, we generated inducible cardiac myocyte-specific RAMP2−/− (C-RAMP2−/−). C-RAMP2−/− exhibited dilated cardiomyopathy-like heart failure with cardiac dilatation and myofibril disruption. C-RAMP2−/− hearts also showed changes in mitochondrial structure and downregulation of mitochondria-related genes involved in oxidative phosphorylation, β-oxidation, and reactive oxygen species regulation. Furthermore, the heart failure was preceded by changes in peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis. Metabolome and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) imaging analyses revealed early downregulation of cardiolipin, a mitochondrial membrane-specific lipid. Furthermore, primary-cultured cardiac myocytes from C-RAMP2−/− showed reduced mitochondrial membrane potential and enhanced reactive oxygen species production in a RAMP2 deletion–dependent manner. C-RAMP2−/− showed downregulated activation of cAMP response element binding protein (CREB), one of the main regulators of mitochondria-related genes. These data demonstrate that the AM-RAMP2 system is essential for cardiac metabolism and homeostasis. The AM-RAMP2 system is a promising therapeutic target of heart failure.
Adrenomedullin (AM) was identified as a vasodilating and hypotensive peptide mainly produced by the cardiovascular system.1 AM is primarily secreted by vascular cells, and it is also secreted from kidney, lung, and heart.2 AM functions as a local autocrine/paracrine mediator,3 as well as a circulating hormone,4 exerting natriuretic,5 antioxidative,6,7 and other effects. AM levels in the blood are increased in hypertension,4,8 heart failure,9 and myocardial infarction,10 which suggests its involvement in cardiovascular disease. Homozygotic AM knockout (AM−/−) mice were dead in utero because of abnormal cardiovascular development.11 On the other hand, heterozygotic AM knockout (AM±) mice were apparently normal, although they exhibited higher blood pressure and cardiac hypertrophy when subjected to cardiovascular stress.6,12 Conversely, transgenic mice overexpressing AM have shown lower blood pressure and resistance to various forms of organ damage,13,14 suggesting that AM exerts organ protective as well as hypotensive effects.15–18
The AM receptor calcitonin receptor-like receptor is a 7-transmembrane domain G protein–coupled receptor19 that associates with an accessory protein, receptor activity-modifying protein (RAMP), which is composed of ≈160 amino acids and includes a single membrane-spanning domain. So far, 3 RAMP isoforms have been identified, but only RAMP2 homozygotic knockout (RAMP2−/−) is lethal in utero and reproduces the phenotypes observed in AM−/− mice.20 This suggests that AM signaling via the calcitonin receptor-like receptor/RAMP2 heterodimer (AM-RAMP2 system) is particularly important for the cardiovascular system. In a rat hypertension model induced by inhibition of nitric oxide synthase, AM and the AM receptor components were reported to be upregulated in the heart, probably as a compensatory response against cardiac hypertrophy.21 Although AM and RAMP2 are highly expressed in the heart from embryo to adulthood, analysis of the pathophysiological functions has been limited by the lethality of both the AM−/− and RAMP2−/− genotypes. We speculated that we can overcome this limitation by using cardiac myocyte-specific conditional gene targeting. For this study, we generated an inducible cardiac myocyte-specific RAMP2 knockout (C-RAMP2−/−) mouse, which enabled us to induce RAMP2 gene deletion in the adult and to directly assess the involvement of the AM-RAMP2 system in the pathophysiology of cardiovascular disease.
For experimental procedures not described herein, please refer to the online-only Data Supplement Methods section.
A mouse line exhibiting cardiac myocyte-specific RAMP2 deletion was generated by cross-breeding RAMP2 flox mice20 with α-myosin heavy chain (MHC)-MerCreMer transgenic (Tg) mice.22 To induce Cre recombination, RAMP2 flox/flox-αMHC-MerCreMer Tg/+ mice (male, 8–12 weeks old) were administered tamoxifen (Sigma) IP once a day for 5 days at a dose of 30 mg/kg per day followed by a 2-day interval without drug administration. For the study of chronic stage after the gene deletion, the treatment was followed by a 23-day interval without drug administration.
All of the experiments were performed in accordance with the Declaration of Helsinki and were approved by the Shinshu University Ethics Committee for Animal Experiments.
Isolation of Embryonic Cardiac Myocytes
Embryonic cardiac myocytes were isolated from embryos at E14.5 to E16.5. Cardiac myocytes from RAMP2 flox/flox-αMHC-MerCreMer Tg/+ embryos or αMHC-MerCreMer (without loxP) embryos were treated with 500 nmol/L of 4-OH-tamoxifen (SIGMA).
Isolation of Adult Cardiac Myocytes
Adult cardiac myocytes were isolated from RAMP2 flox/flox-αMHC-MerCreMer Tg/+ mice treated with tamoxifen or vehicle. Ventricular myocytes were enzymatically isolated from the heart of adult mice, as described previously.23
Ca2+ imaging was performed as described previously.24 Fluorescence images were acquired with a laser scanning microscopy 7 LIVE laser scanning microscope (Zeiss). Ca2+ transient was assessed from fluorescence changes in individual cardiac myocytes in the presence or absence of 10–7 mol/L AM.
Mitochondrial membrane potential was assessed using fluorescent tetramethylrhodamine ethyl ester (TMRE; Molecular Probes) staining. Cardiac myocytes were stained with 100 nmol/L of TMRE for 15 minutes, after which the culture medium was replaced with fresh medium. Mitochondrial reactive oxygen species (ROS) production was assessed using MitoSOX Red (Molecular Probes) staining. The cells were then visualized using a Zeiss laser scanning microscopy 5 EXCITER fluorescence microscope system.
Analysis of Phospholipid Molecular Species
Phospholipids such as cardiolipin were extracted by the Bligh and Dyer method25 and analyzed according to the online-only Data Supplement Methods section.
On-Tissue Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry Imaging Analysis
A frozen section of the heart was placed on an indium tin oxide coated glass slide (578274; Sigma-Aldrich) and stored at −80°C. 9-Aminoacridine at a concentration of 5 mg/mL dissolved in 70% ethanol was used for matrix. Matrix deposition was performed by a chemical printer (CHIP-1000, Shimadzu Corporation) and dried up. Matrix-assisted laser desorption/ionization mass spectra were acquired using AXIMA Performance (Shimadzu) with a 337-nm nitrogen laser, operating in the reflectron/negative mode.
Quantitative values are expressed as the mean±SE. Student t test was used to determine significant differences between 2 groups. One-way ANOVA followed by Fisher protected least significant difference was used to determine significant differences between 3 groups. Dunnett test was used to determine significant differences between >3 groups. Values of P<0.05 were considered significant.
C-RAMP2−/− Mice Exhibit Dilated Cardiomyopathy
To induce cardiac myocyte-specific deletion of RAMP2, RAMP2 flox/flox-αMHC-MerCreMer Tg mice were treated with tamoxifen. It has been reported that a high oral dose of tamoxifen (80 mg/kg body weight for 7 days; total dose, 560 mg/kg) can induce MerCreMer nuclear translocation and dilated cardiomyopathy in mice.22 To avoid the spontaneous occurrence of heart failure, we used a lower dose of tamoxifen (30 mg/kg body weight administered IP daily for 5 days; total dose, 150 mg/kg) and withdrawal for 2 days. After this procedure, the level of RAMP2 gene expression within whole-heart specimens (including both myocytes and nonmyocytes) was reduced to ≈60% of control. We designated tamoxifen-treated RAMP2 flox/flox-αMHC-MerCreMer Tg mice as C-RAMP2−/−. We also confirmed that this dosage does not cause spontaneous heart failure in αMHC-MerCreMer Tg mice without the RAMP2-flox region (Figure S1 in the online-only Data Supplement).
C-RAMP2−/− hearts showed enlargement of ventricles (Figure 1A), with significant increases in the cardiothoracic ratio and heart weight/body weight ratio, although blood pressure was reduced (Figure 1B and 1C). Echocardiography showed C-RAMP2−/− hearts to have dilated left ventricles with diminished systolic function (Figure 1D and Table 1), which suggests that C-RAMP2−/− mice experience dilated cardiomyopathy-like heart failure. Histological analysis revealed enlargement of the cardiac myocytes (Figure 1E and 1G). Electron microscopic observation revealed myofibril disarray and Z-line dislocation, as well as enhanced perivascular fibrosis (Figure 1F). In addition, quantitative reverse-transcription polymerase chain reaction (Figure 1H) showed that brain natriuretic peptide (BNP) and sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) expressions were upregulated and downregulated, respectively, in C-RAMP2−/− hearts. These alterations in gene expression are consistent with the dilated cardiomyopathy-like changes in C-RAMP2−/−.
We further analyzed the chronic phase of the gene deletion. We analyzed C-RAMP2−/− at 23 days after tamoxifen administration. At this stage, C-RAMP2−/− still showed dilatation of ventricles (Figure 2A) with enlargement of cardiac myocytes (Figure 2B and 2C). Echocardiography showed partial recovery of cardiac function; however, compared with control mice, C-RAMP2−/− still showed significantly reduced systolic function with left ventricular dilatation (Figure 2D and Table 2).
Increased Oxidative Stress in C-RAMP2−/− Hearts
We next attempted to identify the cause of the heart failure in C-RAMP2−/−. Immunostaining of 4-hydroxynonenal (HNE), a peroxidized lipid, showed that levels of oxidative stress were elevated in C-RAMP2−/− hearts (Figure 3A, center).
We orally administered the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) to analyze whether the elevated ROS is the primary cause of heart failure in C-RAMP2−/−. TEMPOL treatment reduced ROS levels in the hearts of C-RAMP2−/− (Figure 3A, right). Moreover, the enlargement of cardiac myocytes was reversed (Figure 3B). On the other hand, the altered expression of BNP and SERCA2 was unaffected (Figure 3C), which suggests that mechanisms other than ROS elevation are also involved in the onset of failure in C-RAMP2−/− hearts.
Downregulation of Mitochondrial Membrane-Specific Lipid in C-RAMP2−/− Hearts
To further clarify the mechanisms involved in the failure of C-RAMP2−/− hearts, we used metabolome analysis to comprehensively evaluate the metabolic changes in C-RAMP2−/− hearts. The lipid analysis revealed a distribution shift from phosphatidylcholines to lysophosphatidylcholines (data not shown). Lysophosphatidylcholines derive from the partial degradation of phosphatidylcholines through the removal of 1 of their 2 fatty acids. We speculated that the enhanced oxidative stress in C-RAMP2−/− hearts might promote the degradation of phospholipids.
Furthermore, we found that several forms of cardiolipin were downregulated in C-RAMP2−/− hearts (Figure 4A). Cardiolipin is a mitochondrial inner membrane-specific lipid that binds to cytochrome C, anchoring it in the inner mitochondrial membrane, and is essential for ATP production. To confirm that this downregulation was a result of myocyte abnormalities, we performed matrix-assisted laser desorption/ionization-Time-of-flight mass spectrometry imaging analysis, which enabled us to analyze the metabolome directly using ionized cells from frozen sections and to exclude fibrotic areas and blood vessels. Using this approach, we confirmed that cardiolipins were downregulated in myocytes (Figure 4B). Cardiolipins containing docosahexaenoic acid were downregulated beginning early (day 2) after RAMP2 deletion (Figure 4C). Taken together, these findings suggest mitochondrial changes involving the abnormal distribution of cardiolipin are the earliest events in the heart failure seen after RAMP2 deletion.
Structural and Functional Mitochondrial Abnormalities in C-RAMP2−/− Hearts
Consistent with the results above, electron microscopic observation showed the presence of mitochondrial changes after RAMP2 deletion (Figure 5A). The mitochondrial cristae formations were disrupted. In addition, mitophagy, the phenomena of removing damaged mitochondria, was observed in C-RAMP2−/− hearts (Figure 5A, right). The mitochondrial DNA content was not changed in C-RAMP2−/− hearts (Figure 5B), but quantitative reverse-transcription polymerase chain reaction showed that expression of mitochondria-related genes, including peroxisome proliferator-activated receptor-γ coactivator-1α, β (PGC-1α, β), peroxisome proliferator-activated receptor-α, estrogen-related receptor-α (in charge of mitochondria regulation), carnitine palmitoyl transferase 1α, 2, medium-chain acyl dehydrogenase (in charge of β-oxidation), ATP synthase F1 complex-β subunit, cytochrome C oxidase (in charge of oxidative phosphorylation), uncoupling protein 3, and superoxide dismutase 2 (in charge of mitochondria ROS regulation), were all downregulated in C-RAMP2−/− hearts (Figure 5C). This suggests that heart failure in C-RAMP2−/− is related to mitochondrial dysfunction rather than mitochondrial volume.
We next examined the time course of the gene expression in C-RAMP2−/− hearts. PGC-1α is one of the master regulators of mitochondrial biogenesis. Although we detected no cardiac dysfunction during the period of tamoxifen treatment (from day 0 to 5), gene expression of PGC-1α was transiently upregulated on day 3 and downregulated thereafter (Figure 5D). On the other hand, BNP expression was unaffected during days 0 to 5, and it was upregulated from day 7 onward. This suggests that mitochondrial changes occur before the development of heart failure.
In Vitro Analysis of Cardiac Myocytes From C-RAMP2−/−
To further confirm that mitochondrial dysfunction is the primary cause of heart failure but not secondary to the contractile dysfunction, we cultured primary cardiac myocytes isolated from RAMP2 flox/flox-αMHC-MerCreMer Tg embryos. We then treated the cells with 4-OH-tamoxifen and directly analyzed mitochondrial function. 4-OH-tamoxifen-treated C-RAMP2−/− myocytes showed weak TMRE staining, which indicates depolarization of the mitochondrial membrane (Figure 6A). In addition, Mito SOX red staining revealed mitochondrial ROS levels to be elevated in C-RAMP2−/− myocytes (Figure 6B). We also confirmed that 4-OH-tamoxifen treatment itself did not affect mitochondrial function, because 4-OH-tamoxifen-treated αMHC-MerCreMer without loxP cells showed no changes in TMRE or Mito SOX Red staining (right panels of Figure 6A and 6B). Gene expression of the mitochondrial regulatory factors PGC-1α and β was also suppressed in the C-RAMP2−/− myocytes (Figure 6C).
Furthermore, we analyzed adult cardiac myocytes. In TMRE and Mito SOX Red staining, cardiac myocytes isolated from C-RAMP2−/− showed weaker mitochondrial membrane potential and increased mitochondrial ROS production (Figure 6D). Next, to clarify robustness of the adult cardiac myocytes, we analyzed the Ca2+ transient of the isolated adult myocytes (transient cytosol Ca2+ elevation in response to electric pacing) using a Ca2+ imaging system. Peak twitch amplitude of the cytosol Ca2+ fluorescent after the pacing was significantly lower in C-RAMP2−/− compared with control (Figure 6E). In addition, the decay speed of the transiently elevated Ca2+ fluorescent was slowed, and the half-life of the decay tended to be longer in C-RAMP2−/− than control (Figure 6F). These results suggest that failing cardiac myocytes of C-RAMP2−/− exhibited abnormal Ca2+ handling. With AM stimulation, the amplitude of the Ca2+ transient was elevated in control cardiac myocytes; however, the response was blunted in C-RAMP2−/− myocytes (Figure 6G).
CREB Activation Is Suppressed in C-RAMP2−/− Hearts
AM was originally identified through its ability to increase intracellular cAMP levels. Therefore, to assess signaling pathways involved in the mitochondrial abnormalities in C-RAMP2−/− hearts, we first analyzed intracellular cAMP levels in cultured myocytes from C-RAMP2−/− hearts. cAMP was reduced in C-RAMP2−/− myocytes (Figure 7A). Moreover, Western blot analysis showed that the level of cAMP response element binding (CREB) protein activation was significantly reduced in C-RAMP2−/− hearts (Figure 7B and 7C).
Forskolin stimulates adenylate cyclase directly, thereby increasing intracellular cAMP levels. In C-RAMP2−/− hearts, forskolin treatment partially recovered CREB phosphorylation (data not shown) and mediated repolarization of the mitochondrial membrane in cardiac myocytes (Figure 7D). When we tested its effects in vivo, we found that forskolin reduced ROS levels in C-RAMP2−/− hearts (Figure 7E). In addition, forskolin partially recovered the heart failure-related changes in BNP and sarcoendoplasmic reticulum calcium ATPase 2 expression (Figure 7F). Gene expression of various mitochondria-related factors was downregulated in C-RAMP2−/− compared with control. However, with forskolin treatment, some factors that were related to mitochondria regulation (PGC-1α, PGC-1β, peroxisome proliferator-activated receptor -α, and estrogen-related receptor-α) were significantly upregulated in C-RAMP2−/−. Other factors (CPT-2, medium-chain acyl dehydrogenase, ATP synthase F1 complex β subunit, cytochrome C oxidase, uncoupling protein 3, and superoxide dismutase 2) were also partially restored (Figure 7G).
AM is a circulating peptide and its level in blood is increased in heart failure,9 perhaps as a compensatory response. Although it is known that AM is upregulated in response to various stresses, it is difficult to evaluate the role of AM within the heart using conventional knockout mice, because hemodynamic changes caused by systemic AM deficiency would likely affect cardiac function. Conditional gene targeting is a novel way to solve this problem; however, cardiomyocyte-specific AM knockout mice are also problematic, because AM is secreted from both myocytes and nonmyocytes in the heart, and the secreted AM may work between each other. In the present study, therefore, we chose to disrupt the AM receptor system in cardiac myocytes by generating an inducible conditional RAMP2 gene-targeted mouse.
Figure 8 summarizes our findings for the AM-RAMP system in cardiac homeostasis. We found that cardiomyocyte-specific RAMP2 deletion evoked dilated cardiomyopathy-like heart failure with cardiac dilatation in the adult heart. This heart failure was associated with high levels of oxidative stress. In previous studies using AM± mice, AM was shown to exert strong antioxidant effects in the heart through activation of endothelial nitric oxide synthase and regulation of NADPH oxidase.6,7 As expected, C-RAMP2−/− hearts showed increased oxidative stress, and the superoxide dismutase mimic TEMPOL reversed the increases in ROS. However, because the heart failure caused by RAMP2 deletion was not fully reversed by the ROS reduction, we speculated that other critical factors were involved.
The mitochondria in C-RAMP2−/− hearts showed structural changes that were associated with reduced expression of various mitochondria-related molecules. Furthermore, lipid metabolome analysis and matrix-assisted laser desorption/ionization-time-of-flight mass (MALDI-TOF-MS) spectrometry imaging analysis, which are novel strategies enabling clarification of tissue-specific metabolic alterations, revealed that levels of cardiolipin, a mitochondrial inner membrane-specific lipid, were decreased beginning early after RAMP-2 deletion. Cardiolipin binds to enzymes involved in oxidative phosphorylation, anchoring them to the mitochondrial inner membrane,26 and regulating their activity.27 It is essential for mitochondrial function, and dysregulation of cardiolipin induces heart failure.28 To further confirm that mitochondrial dysfunction is the primary cause of heart failure but not secondary to the contractile dysfunction, we cultured primary cardiac myocytes isolated from RAMP2 flox/flox-αMHC-MerCreMer Tg embryos. We then induced RAMP2 deletion in vitro and proved mitochondrial dysfunction and enhanced ROS production occurred in a RAMP2 deletion-dependent manner. We also confirmed poor Ca2+-handling in cardiac myocytes isolated from adult C-RAMP2−/−.
We found that the altered expression of BNP after RAMP2 deletion was preceded by altered expression of the transcriptional coactivator PGC-1α, which governs mitochondrial biology through broad regulation of genes in both the nuclear and mitochondrial genomes. Induction of PGC-1α is regulated by multiple signaling molecules, including p38 mitogen-activated protein kinase,29,30 Ca2+ calcineurin,31 AMP-activated protein kinase,32 Sirt1,33,34 and CREB.35 In the liver, CREB regulates PGC-1α by binding to the cAMP responsive element in the PGC-1α promoter and inducing expression of the gluconeogenic program.35 Downregulation of CREB is associated with aging, hypertension, insulin resistance, and vascular disease.36 Moreover, transgenic mice overexpressing a dominant-negative CREB in their hearts exhibit dilated cardiomyopathy-like heart failure37 with mitochondrial abnormalities.38 These reports suggest that regulation of PGC-1α by CREB plays a pivotal role in cardiovascular homeostasis. In the present study, we found that CREB activation was reduced in C-RAMP2−/− hearts and speculated that CREB suppression underlies the observed mitochondrial dysfunction and heart failure. Downstream effects mediated by the AM-RAMP2 system include cAMP production, Ca2+ mobilization,39 and activation of Akt and other signaling molecules.40 All of these molecules are involved in CREB activation,41–43 suggesting that RAMP2 deletion suppresses CREB by altering several signaling pathways.
Consistent with that idea, forskolin, which directly stimulates adenylyl cyclase and cAMP production, reversed the mitochondrial membrane depolarization and mitochondrial ROS elevation seen in cultures of primary C-RAMP2−/− cardiomyocytes. In vivo, forskolin ameliorated the oxidative stress and fibrosis in C-RAMP2−/− hearts, reversed the heart failure-related changes in BNP and SERCA2 expression, and restored expression of PGC-1α, estrogen-related receptor-α, (ERR-α) and other mitochondrial genes. These data show that the AM-RAMP2 system is essential for appropriate regulation of cardiac function and homeostasis and that it acts via CREB and PGC-1 to affect mitochondrial ATP production, lipid metabolism, and ROS regulation.
AM is a peptide originally found as a vasodilating and hypotensive peptide secreted from various cells and tissues, including the cardiovascular system. In this study, we directly assessed the roles of the AM-RAMP2 system in cardiac myocytes and its pathophysiological significance in heart failure. These data show that the AM-RAMP2 system may be a promising therapeutic target of heart failure. On the other hand, the clinical applicability of AM, like that of other bioactive endogenous peptides, has 2 serious limitations: AM has a very short half-life in the blood, and the cost of the recombinant protein is very high, which together make the use of AM impractical for treatment of chronic diseases. It is, therefore, noteworthy that we are able to affect the cardiac activity of AM by modulating RAMP2. In fact, we would expect that greater specificity can be achieved by targeting RAMP2 than by targeting calcitonin receptor-like receptor, which can also function as a receptor for α-calcitonin gene-related peptide, β-calcitonin gene-related peptide, amylin, and intermedin. In this study, we have shown that the AM-RAMP2 system is essential for cardiac homeostasis. Our findings could potentially provide the critical basis for developing medicines targeting the AM-RAMP2 system, which could directly promote both energy production and ROS suppression in cardiac myocytes.
Sources of Funding
This study was supported by the Funding Program for
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Novelty and Significance
What Is New?
By generating cardiac myocyte-specific RAMP2 deletion model mouse (C-RAMP2−/−), we directly proved the critical roles of AM-RAMP2 system in the heart.
C-RAMP2−/− developed dilated cardiomyopathy-like heart failure.
We found novel regulation of cardiac mitochondrial function by the AM-RAMP2 system.
What Is Relevant?
AM is a promising therapeutic molecule for treating hypertensive diseases through its well-known vasodilative function. In this study, we proved that the AM-RAMP2 system also directly regulates cardiac metabolism and homeostasis.
AM-RAMP2 system is a promising therapeutic target for heart failure in addition to hypertension.