Tumor Necrosis Factor-α–Converting Enzyme Is a Key Regulator of Agonist-Induced Cardiac Hypertrophy and Fibrosis
Cardiac remodeling is associated with hypertrophy and fibrosis processes, which may depend on the activity of matrix metalloproteinases (MMPs) and “a disintegrin and metalloproteinases” (ADAMs). We investigated whether ADAM-17 (tumor necrosis factor-α–converting enzyme [TACE]) plays a role in agonist-induced cardiac remodeling and the relationships established among TACE, MMP-2, and ADAM-12. We targeted TACE in rodent models of spontaneous and agonist-induced hypertension using RNA interference combined with quantitative RT-PCR, activity determinations, and functional studies. Treatment of spontaneously hypertensive rats with previously validated TACE small-interfering RNA for 28 days resulted in systemic knockdown of TACE expression. TACE knockdown effectively stopped the development of cardiac hypertrophy. Mice receiving angiotensin II (1.4 mg/kg per day for 12 days) exhibited cardiac hypertrophy, as well as fibrosis, which was associated with elevated myocardial expression of molecular markers of hypertrophy (α-skeletal actin, β-myosin heavy chain, and brain natriuretic peptide) and fibrosis (collagen types I and III and fibronectin), as well as MMP-2 and ADAM-12. Treatment with TACE small-interfering RNA (but not with PBS or luciferase small-interfering RNA) inhibited TACE expression, thus preventing angiotensin II–induced cardiac hypertrophy and fibrosis. Moreover, knockdown of TACE inhibited angiotensin II–induced overexpression of markers of myocardial hypertrophy and fibrosis, as well as ADAM-12 and MMP-2. These findings provide the first in vivo evidence that agonist-induced cardiac hypertrophy and fibrosis processes are signaled through TACE, which acts through novel pathways involving transcriptional regulation of ADAM-12 and MMP-2. Targeting TACE has potential therapeutic importance for modulating agonist-induced cardiac remodeling.
- cardiac hypertrophy
- Gq protein–coupled receptor agonist
- RNA interference
Cardiac remodeling is a major hallmark of hypertensive disorders and is associated with the development of cardiac hypertrophy (ie, an increase in cell size of individual cardiomyocytes), which causes thickening of the myocardium. Although initially compensatory, sustained hypertrophic growth is pathological, in part, because of its association with the development of fibrosis (ie, increased synthesis and deposition of extracellular matrix proteins), which disrupts the normal structure and contractile properties of the myocardium.1 Pathological cardiac remodeling is, thus, detrimental for cardiac function and may cause cardiac dysfunction, myocardial stiffness, and increased risk of heart failure, sudden death, and stroke.2–5 However, it remains unclear how and why such apparently distinct processes as cardiac hypertrophy and fibrosis develop with hypertension and whether pressure overload in hypertension is causal.
Our laboratory is investigating the general hypothesis that cardiac remodeling may be associated with hypertension simply because high blood pressure, hypertrophy, and fibrosis share common inducers, which signal through largely overlapping pathways. Among the common inducers of high blood pressure, hypertrophy, and fibrosis are vasoconstrictive agonists, eg, catecholamines, endothelins, and angiotensin (Ang) II.6–9 These agonists all act on Gq protein–coupled receptors, which, in turn, activate the classic phospholipase C/protein kinase C pathway and reactive oxygen species, leading to the activation of downstream matrix metalloproteinases (MMPs) and “a disintegrin and metalloproteinases” (ADAMs). Among them, ADAM-12 and ADAM-17 (tumor necrosis factor-α–converting enzyme [TACE]) are perhaps the best studied in the cardiovascular and endocrine systems.7–12 ADAM-12 and TACE are synthesized and stored in the rough endoplasmic reticulum until they mature in a late Golgi compartment. Their maturation after agonist stimulation involves the removal of the prodomain from the precursor protein. The activated metalloproteinases from both MMP and ADAM families are able to cleave a host of common substrates, including extracellular matrix (ECM) proteins (eg, collagens), proinflammatory mediators (eg, tumor necrosis factor-α), and growth factors (eg, transforming growth factor-α and heparin-binding epidermal growth factor–like growth factor), to signal through their receptors and downstream mitogen-activated protein kinases, which transcriptionally activate the expression of immediate-early genes and fetal genes, including hypertrophy markers.13 There is increasing evidence that different metalloproteinases, including MMP-2, MMP-7, ADAM-12, and TACE, may, thus, mediate tissue remodeling and injury in both cardiovascular and renal systems.7,9,12,14–18
The similar tissue localization, agonist-activation profile, substrates, and signaling pathways of some of the growth factor sheddases, eg, ADAM-12 and TACE, suggest a redundancy of their functions in vivo, particularly in signaling of cardiovascular growth processes.7–9,12,19,20 ADAM-12 has previously been directly implicated in cardiac hypertrophy.7 However, the involvement of TACE in cardiac hypertrophy has been suggested9,10 but not yet demonstrated in vivo. Although TACE is a key mediator of Ang II–induced renal injury and fibrosis,9,10,21 it is unknown whether TACE mediates the development of agonist-induced cardiac fibrosis.
If TACE plays a role in the development of cardiac hypertrophy and fibrosis, it would be important to determine whether and, if so, how TACE and ADAM-12 coordinate each other’s expression and functional redundancy. Would their relationships follow a hierarchical pattern? Would such hierarchical relationships be of significance for the mechanisms and treatment of pathological cardiac remodeling?
Here we start to address these important questions by focusing on the role of TACE in the development of agonist-induced cardiac hypertrophy and fibrosis processes. Our findings establish a novel central role for TACE in signaling of both processes, upstream of MMP-2 and ADAM-12.
Materials and Methods
Please see the online Data Supplement (available at http://hyper.ahajournals.org) for the expanded Methods section.
Generation of TACE Knockdown Models Using Small-Interfering RNA
The animal (mouse and rat) model of TACE expression knockdown was generated using a previously validated TACE small-interfering RNA (siRNA)10 (for siRNA design, please see Figure 1) synthesized by Sigma-Aldrich. Luciferase (Luc) siRNA (antisense: 5′-GUAUCUCUUCAUAGCCUUAdTdT) was used as the control. The first 2 nucleotides of each strand were 2′-O methylated to increase siRNA stability. The siRNAs were dissolved in PBS before use.
siRNA Studies in Rats
siRNA (9 nmol/kg per day, ie, 0.12 mg/kg per day) or PBS was infused for 14 days into spontaneously hypertensive rats (SHRs) through subcutaneously implanted ALZET osmotic minipumps (DURECT Corporation) in the back of the animals.
siRNA Studies in Mice
siRNA (30 nmol/kg per day, ie, 0.4 mg/kg per day) or PBS was infused for 14 days into C57bl/6J mice through subcutaneously implanted osmotic minipumps. Another group of mice received siRNA (15 μg per mouse, ie, 0.45 mg/kg) or PBS by injection via the jugular vein following a recently described method.22 The injection was conducted every 5 days to maintain the knockdown effects.
Mouse Model of Ang II–Induced Hypertension and Cardiac Hypertrophy
Male C57BL/6 mice were infused with Ang II (1.4 mg/kg per day) through subcutaneously implanted osmotic minipumps for 12 days.
RNA Expression Analysis by TaqMan RT-PCR
Total RNA was extracted from flash-frozen tissue using TRIzol (Invitrogen), and cDNA was generated from 1 μg of RNA using a random hexamer. Expression analysis of the reported genes was performed by TaqMan RT-PCR using the ABI 7900 sequence detection system. 18S rRNA was used as an endogenous control, as described previously.23
TACE Mediates Cardiac Hypertrophy in Genetically Hypertensive Rats
We examined whether blocking TACE expression in already-hypertensive 22-week–old SHRs would affect systolic blood pressure and/or development of cardiac hypertrophy. To inhibit TACE gene expression, we chose an RNA interference–based approach using a previously validated siRNA10 with the exception that we introduced a chemical modification (2′-O methylation) on the 5′ end of the double-stranded RNA molecule to enhance its resistance to nucleases in vivo (Figure 1). The administration of TACE siRNA for 30 days through a subcutaneous osmotic minipump (protocol depicted in Figure 2A) effectively stopped the progression of cardiac hypertrophy, as evidenced by M-mode echocardiography and gross pathology studies (Figure 2B and 2C and Table 1). In addition, the cross-sectional width of cardiomyocytes was decreased on average by 38% in SHRs receiving TACE siRNA versus SHRs receiving vehicle (PBS; n=3 rats in each study group; P<0.05 by t test), confirming the antihypertrophic effects of TACE siRNA.
Rats receiving TACE siRNA had significantly lower TACE immunoreactivity and TACE proteolytic activity (Figures 2D and S1 and data not shown), MMP-2 activity (Figure 2D), and extracellular signal–regulated kinase 1/2 phosphorylation (Figure S2) compared with rats receiving an unrelated RNA or vehicle (PBS). These data indicated that the TACE siRNA effectively decreases TACE expression and activity, as well as TACE downstream signaling. Interestingly, treatment with TACE siRNA did not decrease the blood pressure in the SHR model (Figure 3A) despite TACE siRNA causing a significant inhibition of TACE proteolytic activity in resistance arteries of the rats (Figure 3B). Taken together, these observations provide strong in vivo evidence that TACE expression regulates development of cardiac hypertrophy, but TACE may not play a major role in the regulation of blood pressure in hypertension.
TACE and ADAM-12 Form a Novel Signaling Axis In Vivo
To further clarify how TACE knockdown impacts the development of cardiac hypertrophy, we conducted functional studies, quantitative RT-PCR, and activity determinations in mice where TACE was knocked down by in vivo RNA interference. Administration of TACE siRNA (0.4 mg/kg per day) through subcutaneous osmotic minipumps for 14 days in mice resulted in a significant downregulation in myocardial TACE mRNA levels (Figure 4A) and proteolytic activity (Figure 4B).
To substantiate the effects of TACE siRNA, we conducted a small additional study (n=2 mice per group) using a different route of siRNA administration (ie, intravenous). Mice were injected with TACE siRNA (15 μg every 5 days) into their jugular vein, a method that was shown to effectively knock down the expression of cardiac target proteins.22 TACE expression was decreased by 30±5% in mice that received TACE siRNA by intravenous injection versus mice that received PBS. Mice that received TACE siRNA did not display any echocardiographic abnormalities (Table 2).
Interestingly, knockdown of TACE resulted in the downregulation of MMP-2 mRNA levels (Figure 4C) and activity (Figure 4D), as well as in ADAM-12 gene expression (Figure 4E). However, the siRNA had otherwise insignificant effects on other genes, including MMP-9, tissue inhibitor of metalloproteinase 3 (an endogenous inhibitor of TACE20), and interferon-γ (data not shown; n=4 mice; P>0.05 by t test). In mice receiving siRNA by jugular vein injection, the level of interferon-γ was also unaffected (data not shown). The unchanged level of interferon-γ demonstrated that the small RNA-induced innate immune response24 was not activated. This further suggested that the observed downregulation of MMP-2 or ADAM-12 was caused by a decrease in TACE expression rather than a nonspecific effect of the TACE siRNA.
As expected, the blood pressure of mice receiving Ang II through osmotic minipumps (1.4 mg/kg per day for 10 days) was significantly elevated versus mice receiving PBS (BP[PBS+Ang II]=188±1 mm Hg; BP[PBS]=133±2 mm Hg; P<0.001 by t test; n=4 mice per study group). Mice receiving either TACE siRNA or Luc siRNA (0.4 mg/kg per day) by osmotic minipumps also developed hypertension on infusion of Ang II for 10 days (BP[TACE siRNA+Ang II]=167±13 mm Hg; BP[Luc siRNA+Ang II]=180±2 mm Hg; n=4 mice per group). Similarly, after 10 days of Ang II infusion, blood pressure was elevated in mice receiving PBS, TACE siRNA, or Luc siRNA by jugular vein injection (BP[Ang II]=179±4 mm Hg; BP[TACE siRNA+Ang II]=173±3 mm Hg; BP[Luc siRNA+Ang II]=179±3 mm Hg; n=3 to 4 mice per study group). Interestingly, in mice that received TACE siRNA by intravenous injection (but not in those that received TACE siRNA by osmotic minipumps) there seemed to be a delay in the onset of the hypertension. Indeed, after 5 days of Ang II infusion, blood pressure was higher in mice that received either PBS or Luc siRNA by intravenous injection versus those that received TACE siRNA (BP[PBS+Ang II]=150±3 mm Hg; BP[Luc siRNA+Ang II]=164±9 mm Hg; BP[TACE siRNA+Ang II]=122±11 mm Hg; n=3 to 4 mice per study group).
Mice that received Ang II (1.4 mg/kg per day) for 12 days displayed elevated expression and proteolytic activity of TACE, MMP-2, and ADAM-12 (Figures 4 and S3), as well as left ventricular hypertrophy (Table 2 and Figures 5, 6⇓, and S4), which was associated with an overexpression of myocardial hypertrophy marker genes (brain natriuretic peptide and α-skeletal actin and β-myosin heavy chain; Figures 5, 6⇓, and S4). Cardiac fibrosis, the increased deposition of ECM proteins in myocardium that is typically associated with pathological cardiac hypertrophy, was also induced by Ang II, as shown by the increased expression of the ECM proteins, collagen types I and III, and fibronectin (Figures 5, 6⇓, and S4).
Pretreatment with TACE siRNA through osmotic minipumps decreased cardiac TACE levels and activity (Figure 4) and fully protected the mice from Ang II–induced left ventricular hypertrophy and cardiac fibrosis (Table 2 and Figure 5). The protective effect of TACE siRNA was associated with the normalization of the expression of MMP-2, ADAM-12 (Figure 4), hypertrophy marker genes, and ECM proteins (Figure 5). Unlike TACE siRNA, the treatment of mice with an siRNA against Luc did not protect from Ang II–induced left ventricular hypertrophy and fibrosis, as evidenced by M-mode echocardiography, gross pathology studies, and RT-PCR analysis of molecular markers of hypertrophy and fibrosis (Figure 6).
Furthermore, excluding a role of the administration route in the protective effects of TACE siRNA, in mice receiving siRNA by jugular vein injection, TACE siRNA also blocked the Ang II–induced expression of TACE (Figure S3A), MMP-2 (Figure S3B), and ADAM-12 (Figure S3C). TACE siRNA, but not Luc siRNA, prevented Ang II–induced cardiac hypertrophy, as indicated by gross pathology (Figure S4A), cardiomyocyte cross-sectional area (Figure S4B), and expression of hypertrophic markers (Figure S4C). In addition, knockdown of TACE by siRNA also protected mice from Ang II–induced cardiac fibrosis, as determined by cardiac interstitial collagen staining with Picrosirius red (Figure S4D) and expression of collagen I (Figure S4E).
These findings are the first evidence that the downregulation of TACE expression may prevent the transcription of metalloproteinases, eg, MMP-2 and ADAM-12, and downstream hypertrophy markers, which together could act as effectors of TACE in agonist-induced cardiac hypertrophy.
This investigation has resulted in several novel observations. First, to our knowledge, our findings suggest for the first time in vivo that agonist-induced cardiac hypertrophy and fibrosis are signaled through ADAM-17/TACE, and decreasing TACE activity by RNA interference protects from cardiac hypertrophy and fibrosis in models of hypertension. Furthermore, our data suggest that TACE may act by promoting the transcription of metalloproteinases (eg, MMP-2 and ADAM-12) and downstream genes of so-called molecular markers of hypertrophy and fibrosis. Together, these pathways likely act as effectors of cardiac remodeling downstream of ADAM-17/TACE. Moreover, the findings indicate that targeting TACE disrupts signaling through MMP-2 and ADAM-12, which could have generic therapeutic value for therapeutic management of cardiac hypertrophy and fibrosis, 2 processes that invariably develop subsequent to sustained vasoconstrictive agonist stimulation.5
A critical assessment of the data gathered in this research would suggest that the protective effects of TACE siRNA in agonist-induced cardiac hypertrophy (in Ang II–infused mice, as well as SHR models) were primarily attributed to the downregulation of TACE. In all of the models, the degree of TACE knockdown, albeit modest, was significant, ≈40% from baseline measured by either immunoreactivity or proteolytic activity and 25% as measured by quantitative RT-PCR. Potential off-target effects, eg, signaling through the toll-like receptor/interferon-γ pathway,24 may not be major mechanisms of protective effects of TACE siRNA because administration of an siRNA to a nonmammalian gene (Luc) by 2 different routes of administration (ie, infusion through minipump and injection via jugular vein) had no protective effect on Ang II–induced hypertension, cardiac hypertrophy, and fibrosis, and no upregulation of interferon-γ was observed; these in vivo findings are in agreement with a recent report using the same siRNA sequence on myoblast cultures.10
TACE siRNA had no long-term protective effect on hypertension in either SHRs or Ang II–infused mice. However, we cannot exclude that TACE siRNA treatment could have a short-term or transient protective effect that was nonetheless insufficient to prevent the development of hypertension in both SHRs and Ang II-infused mice. Because of the complexity of the effects of TACE inhibition on the transcription of multiple genes, dedicated studies are warranted to further dissect the mechanism of the short-term roles of TACE in the regulation of blood pressure of hypertension. However, our long-term data are consistent with previous research showing that the pharmacological inhibition of TACE (with TAPI-2) does not decrease systolic blood pressure in Ang II–infused mice.12 Our long-term data are also in agreement with previous research showing that pharmacological blockade (with KB-R7785) of ADAM-12 (which we found to be downstream of TACE) does not protect mice from agonist-induced hypertension.7
Previous studies have found that TACE, ADAM-12, and MMP-2 are all upregulated in human hypertrophic cardiomyopathy16,21 and that ADAM-12 may mediate agonist-induced cardiac hypertrophy.7 Recently, a novel role for ADAM-12 was reported in facilitating activation of transforming growth factor-β signaling through Smads, which is the main pathway mediating the development of agonist-induced fibrosis. This action of ADAM-12 is mediated via protein-protein interactions, independent of ADAM-12 protease activity.25 Similarly, MMP-2 has been shown to promote myocardial hypertrophy and interstitial fibrosis through activation and release of transforming growth factor-β,26 thus facilitating Smad signaling.11 Previous work also showed that TACE knockdown by RNA interference blocks mechanotransduction-induced myogenesis in cultured myoblasts10 and that primary cultured aortic vascular smooth muscle cells expressing a dominant-negative mutant of TACE do not develop hypertrophy in response to Ang II.9 Although the potential involvement of TACE in cardiac hypertrophy and fibrosis is suggested by previous research,9,10,21 TACE involvement in these processes has not been demonstrated in vivo.
This study has identified novel and significant effects of TACE siRNA on both cardiac hypertrophy and fibrosis in vivo. Our observations provide the first in vivo evidence that TACE expression regulates the development of cardiac hypertrophy and fibrosis and, as such, substantially expands previous research. Taking our findings together with previous investigations, we suggest that an overabundance of vasoconstrictive agonists (as occurs in hypertensive disorders) could posttranscriptionally enhance the activity of multiple metalloproteinases, eg, TACE and ADAM-12, which next signal through a common pathway. Accordingly, the activated metalloproteinases may act by shedding growth factors and cytokines (eg, heparin-binding epidermal growth factor–like growth factor, transforming growth factor-α, and tumor necrosis factor-α) to promote the transcription of immediate-early, fetal, and ECM genes, which are key mediators of hypertrophy and fibrosis processes7–9,12,19,20 (Figure S5).
Despite extensive research,7–9,12,19,20 an interaction among the pathways of TACE, ADAM-12, and MMP-2 has never been identified. Therefore, a novel finding of this research has been the observation that baseline gene expression levels of TACE and ADAM-12 are transcriptionally connected, although further research is necessary to dissect the transcriptional pathways linking these metalloproteinases. Together with previously reported agonist-induced posttranscriptional signaling events,7–9,12,19,20 the TACE/ADAM-12 signaling axis may regulate the development and progression of hypertrophy and fibrosis, which are hallmarks of agonist-induced cardiovascular remodeling (Figure S5).
Furthermore, it is possible that metalloproteinases form a highly regulated signaling network (as opposed to acting in isolation). Certain metalloproteinases (eg, TACE) may act like primary mediators of cardiovascular (hypertrophic) growth and fibrosis processes, whereas other metalloproteinases (eg, MMP-2 and ADAM-12) are downstream effectors. Interestingly, we found recently that MMP-7 may exert a transcriptional regulation of ADAM-12 in cardiac hypertrophy, similar to that demonstrated here for TACE.27
These current findings have therapeutic potential in hypertensive heart disease. Our data suggest that TACE, MMP-2, and ADAM-12 may define a novel signaling axis in agonist-induced cardiac hypertrophy and fibrosis processes and that these processes can be disrupted by targeting TACE. This notion is supported by our studies both in mice with agonist-induced cardiac hypertrophy and in SHRs, a model where cardiac hypertrophy is likely signaled by multiple agonists.28–30 Together with our previous studies on MMP-7 and ADAM-12, it may be possible to counter different hypertensive cardiac diseases and specific complications thereof by targeting one or more nodes in the emerging network of interconnected metalloproteinases, which includes TACE, MMP-7, MMP-2, and ADAM-12.
Future studies should investigate the emerging notion of metalloproteinase networks as mediators of agonist-induced cardiovascular hypertrophy and fibrosis processes and the dynamics of this network in various models and stages of hypertension and cardiac remodeling.
Sources of Funding
This work was supported by research grants of the Alberta Heritage Foundation for Medical Research (block-grant), the Natural Sciences and Engineering Council and the Canadian Institutes of Health Research (to C.F.-P., who is also a Canadian Institutes of Health Research New Investigator). This work was also supported by Canadian Institutes of Health Research research grants to Z.K. and G.D.L.
X.W. and T.O. contributed equally to this work.
The authors had full access to and take full responsibility for the integrity of the data. All of the authors have read and agree to the article as written.
- Received December 9, 2008.
- Revision received December 31, 2008.
- Accepted June 11, 2009.
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