Targeting Focal Adhesion Kinase With Small Interfering RNA Prevents and Reverses Load-Induced Cardiac Hypertrophy in Mice
Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing (≈70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of β-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.
Left ventricular hypertrophy is a common feature of preclinical cardiovascular diseases with prognostic implications. Although the hypertrophy initially assumes a key role in the cardiac compensation for hemodynamic overload, it often evolves with structural and functional abnormalities that predispose to chamber maladaptive remodeling, heart failure and premature death.1,2 Early hypertrophic growth reflects the increased mass of cardiac myocytes, whereas maladaptive remodeling is additionally accompanied by abnormalities such as disproportionate growth of the myocardial interstitium, intrinsic dysfunction, and death of the myocytes that are postulated to be the underlying causes of the hypertrophy decompensation.3 Mechanical stress elicited by changes in hemodynamic forces and overactivity of neurohormonal factors have been considered the main triggering stimuli for the installation of hypertrophy as well as of the structural and functional deterioration occurring during hypertrophy decompensation.4,5
Focal adhesion kinase (FAK) has received attention as a potential mediator of the cardiac myocyte responses to hypertrophic stimuli. Accordingly, FAK has been demonstrated to mediate the expression of hypertrophic genetic program in isolated cardiac myocytes in response to mechanical stress6–8 or to agonists such as endothelin and phenylephrine.9–11 Moreover, FAK expression has been shown to be persistently increased in hypertrophic rat and human left ventricle (LVs).12,13 Recently, myocyte-restricted FAK conditional knockout mice were generated by 2 independent groups that confirmed the critical importance of FAK for the load-induced hypertrophy of the LV.14,15 However, these authors reported contrasting phenotypic changes of the LV in response to pressure overload. Peng et al14 reported an eccentric hypertrophy and an increased length of cardiac myocytes, whereas DiMichele et al15 reported an attenuation of hypertrophy in response to pressure overload. These conflicting data and the limitations imposed by restricting the FAK deletion to cardiac myocytes preclude a better understanding of the role of FAK in the complex pathophysiology of cardiac hypertrophy and its progress to failure.
In the present study, we report the development of a strategy to deplete FAK in mouse LVs by in vivo small interfering (si)RNA delivery and to address the influence of FAK in the pathophysiology of cardiac hypertrophy and failure induced by chronic pressure overload in mice. We found that myocardial FAK silencing prevented and reversed left ventricular hypertrophy, myocardial fibrosis, and the deterioration of cardiac structure and function in chronically overloaded LVs.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Swiss mice (6 to 8 weeks old) were handled in compliance with the principles of laboratory animal care formulated by the Animal Care and Use Committee of the State University of Campinas. Procedures such as jugular and tail vein and carotid and femoral artery catheterization, aortic banding, and echocardiographic examination were performed under anesthesia with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg). Chronic pressure overload was obtained by transverse aortic constriction. Blood pressure was simultaneously recorded in the carotid and femoral arteries to determine the transconstriction systolic gradient.
Two-dimensional M-mode echocardiography was performed with a 12-MHz probe connected to a Toshiba Power Vision system by a blinded observer at 15 minutes after the induction of anesthesia.
siRNA targeted to the mouse FAK gene was designed by a software that used internal stability parameters, as previously reported.16 DNA oligonucleotides (Integrated DNA Technologies) were as follows: (1) T7, 5′-GGTAAT ACGACTCACTATAG-3′; (2) FAK sense (AB030035), 5′-GCGAAATCCATAGCAGGCCACTA TAGTGAGTCGTATTACC-3′; (3) FAK antisense, 5′-ACGTGGCCTGCTATGGATTTCTATAGTGAGTCGTATTACC-3′; (4) GFP sense, 5′-GTGTCTTGTAGTTCCCGTCTATAGTGAGTCGTATTACC-3′; (5) GFP antisense, 5′-ATGACGGGAACTACAAACACC-TATAGTGAGTCGTATTACC-3′.
Isolation of Adult Mouse Ventricular Myocytes and Fibroblasts
Ventricular myocytes and fibroblasts were isolated from mice 1 day after treatment with siRNAFAK or siRNAGFP using a collagenase digestion method, as reported previously.17
Tissue extracts containing equal amounts of total protein (50 μg) were resolved by 8% SDS-PAGE. The membranes were incubated with primary antibodies and 125I-protein A was used for the detection of specific bands.
Samples of total RNA (15 μg) were resolved in denaturing gel and blotted with 32P-labeled oligonucleotide FAK probes (151 bp) generated using the following primers: FAK sense (AB030035), 5′-ATGTTCTGGTGTCCTCAAATG-3′ (gene coordinates 1590 to 1610); and FAK antisense, 5′-GAGGTAAAACGTCGAAAATTG-3′ (gene coordinates 1720 to 1740).
Samples of mouse LVs were resolved in SDS-PAGE containing 0.1% of gelatin. The gels were incubated for 24 hours in a matrix metalloproteinase (MMP) substrate buffer and then stained with a Coomassie blue solution. The MMP proteolytic activity was determined by image analysis of the zymograms.
Samples of left ventricular posterior wall were used for quantitative estimation of myocardial hydroxyproline.
Plasma interferon-γ concentration was obtained with an endogen mouse interferon-γ ELISA MiniKit according to the instructions of the manufacturer (Pierce).
Sections of LVs were stained with Masson’s trichrome to measure the cardiac myocyte diameter and the interstitium in heart sections.
Immunohistochemistry was performed as described previously.7
One-step RT-PCR was performed to assess the expression of β-myosin heavy chain (MHC) (sense, 5′-GCCAACACCAACCTGTCCAAGGTTC-3′; and antisense, 5′-TGAAAAGGC TCCAGGTCTGAGGGC-3′) and GAPDH (sense, 5′-GGCATTGCTCTCAATGACAA-3′; antisense, 5′-AGGGTGCAGHGGAACTTTATT3′) was used as the reaction control.
Data are presented as means±SEM. Differences between the mean values of the hemodynamic and echocardiographic data and densitometric readings were tested by 1-way or 2-way repeated-measures ANOVA. Post hoc analysis was performed with Bonferroni multiple-range test. Survival of siRNAFAK versus siRNAGFP banded mice were compared using the Kaplan–Mayer test. A value of P<0.05 indicated statistical significance.
FAK Expression and Phosphorylation Are Increased in Hypertrophic LVs
To examine FAK protein expression and phosphorylation at Tyr397 in the hypertrophic LVs, we studied different subgroups of mice that underwent sham operation (SO) or aortic banding (1 to 12 weeks). As shown in Figure 1A, left ventricular posterior wall thickness was already increased in 1-week banded mice, demonstrating the rapid hypertrophic growth typical of this model. Dilatation and reduction of fractional shortening of the LVs were also observed in this subgroup, likely because of the impact of the sudden aortic constriction. Increased LV wall thickness without dilatation or reduction in fractional shortening, were seen in mice studied 2, 3, or 6 weeks after aortic banding, indicating a compensated hypertrophy. However, 12-week banded mice showed chamber dilatation and reduction in fractional shortening, in addition to hypertrophy, indicating a stage of LV decompensation. Detailed hemodynamic, echocardiographic, and morphometric data of these subgroups are shown in supplemental Table I. Postmortem analysis confirmed the increases of left ventricular mass in banded mice. As expected, the average diameter of cardiac myocytes and the area occupied by the myocardial interstitium were increased in banded mice (supplemental Table I).
Greater amounts of FAK were seen in the LVs of banded in comparison with SO mice (≈2.0-fold in 1- to 12-week banded mice) (Figure 1B). Phosphorylated FAK was also increased in samples of banded mice, peaking at the first and at the 12th week (≈3.5-fold) after aortic banding, so that the ratio of phosphorylated to total FAK (pFAK/FAK) (Figure 1B) was increased by 2.0- and 1.8-fold, respectively. Notably, the peaks in the pFAK/FAK ratio coincided with dilatation and reduced fractional shortening of the LVs. Samples were normalized to the densitometric values of cardiac myosin (Figure 1B). Coomassie staining of total LV protein resolved in SDS-PAGE, confirmed the loading of equal amounts of total myocardial protein in the gels (Figure 1B).
Immunostaining with anti-FAK antibody indicated that in the LVs of SO mice, FAK was mostly located in the cardiac myocytes (Figure 1C and 1D), with only a faint staining of the interstitial areas. However, in samples from banded mice, anti-FAK staining was detected in cardiac myocytes and in the areas of focal and perivascular interstitial fibrosis (Figure 1E and 1F).
Optimization of Myocardial FAK Silencing by siRNA
Next, we searched for an amount of siRNAFAK that, when injected systemically via the jugular vein, could reduce the myocardial FAK protein expression by at least 70% compared with the values normally seen in the mouse LVs. We started with 5 μg (≈150 μg/kg) of siRNA based on previous reports in the literature18,19 and demonstrated that 72% silencing of myocardial FAK could be obtained 24 hours after bolus injections of 15 μg (≈500 μg/kg) of siRNAFAK diluted in 300 μL of PBS via the jugular vein (Figure 2A). We also examined whether high-pressure hydrodynamic injections of siRNAFAK via the tail vein were effective in inducing FAK silencing in the mouse LVs. The representative example of Figure 2B indicates that injections of 15 μg of siRNAFAK, diluted in 3 mL PBS, did not affect FAK expression in the LVs, likely because of the dilution of the siRNA.
The next aim of this study was to investigate the time course of myocardial FAK silencing after single injections of siRNAFAK that were delivered systemically. As summarized in Figure 2C, myocardial FAK protein expression was reduced to ≈28% at 1 and to ≈50% at 7 and 15 days after the administration of siRNAFAK in the LVs of normal mice. By the third week after siRNAFAK injection, the amount of myocardial FAK reached levels comparable to those seen in mice treated with siRNAGFP. The analysis of Northern blots confirmed a similar reduction in FAK mRNA over time after siRNAFAK injection (Figure 2D).
Additional experiments were performed to investigate whether myocardial FAK gene silencing included the cardiac myocytes and the fibroblasts. As shown in Figure 2E and 2F, FAK protein expression was reduced in the extracts from myocytes or fibroblasts harvested from the LVs 24 hours after the treatment with siRNAFAK to an extent comparable to that of myocardial extracts.
Next, we aimed to investigate potential off-target and nonspecific effects related to siRNAFAK. As shown in Figure 3A and 3B, no changes were seen in the myocardial expression of FAK-related protein tyrosine kinase, proline-rich tyrosine kinase 2 (Figure 3A), extracellular signal-regulated kinase ½, or cardiac myosin (Figure 3B) 24 hours after the injections of siRNAFAK in normal mouse LVs. Another potential problem with RNA interference in mammalian systems is the induction of the interferon response, which causes global, nonspecific inhibition of protein translation and might influence the effects of siRNAFAK in the myocardium. As shown in Figure 3C, there was no significant change in the plasma levels of interferon-γ in a period ranging from 1 to 28 days after the injections of siRNAFAK or siRNAGFP in normal mice.
Finally, because the siRNA was injected systemically, we also analyzed FAK protein expression in tissues such as lung, kidney, and liver. Reductions in FAK expression similar to those seen in myocardial samples were observed in the lung and in the kidney; however, no change was detected in the liver after injections of siRNAFAK (Figure 3D). The difference in the efficiency of FAK silencing in the liver and kidney might be related to differences in the arterial blood flow and mass between these organs.
FAK Silencing Retards Load-Induced Hypertrophy in Aortic-Banded Mice
To investigate whether myocardial FAK silencing affects the load-induced LV hypertrophy, siRNAFAK or siRNAGFP were injected on the day before sham operation or aortic banding (1 to 12 weeks). The data shown in Figure 4A indicate that single injections of siRNAFAK significantly reduced FAK protein expression in the LVs of aortic-banded mice. This effect was still observed, although attenuated, in the group of mice studied 2 weeks after the siRNA injection. In contrast, in banded mice injected with siRNAGFP, FAK protein expression was found to be increased at the first week after aortic banding and then thereafter. Next, experiments were performed to examine whether FAK silencing was also reflected in FAK mRNA levels. As shown in Figure 4B, aortic banding lasting for 7 days increased FAK mRNA expression by 2.4-fold in mice treated with siRNAGFP, whereas in mice treated with siRNAFAK, it was found to be reduced by ≈40%. Moreover, as shown in Figure 4C, in 7-day banded mice, the reductions in the myocardial expression of FAK induced by siRNAFAK were accompanied by a proportional reduction in the amount of phosphorylated FAK.
We found that blood pressure, heart rate, and structure and function of LVs were unaffected by siRNAFAK in SO mice, indicating that FAK knockdown does not influence basal cardiac function or structure; thus the data of 6-week SO mice treated with siRNAFAK or siRNAGFP were used as controls for the comparisons with banded mice. Treatment with siRNAGFP did not affect the typical changes of the LV induced by chronic pressure overload (Figure 4D, black bars). Pretreatment with siRNAFAK retarded the load-induced left ventricular hypertrophy, an effect that paralleled the FAK protein silencing. Transient FAK silencing markedly attenuated the dilatation as well as the reduction of left ventricular fractional shortening seen at the first week after aortic banding (Figure 4D, empty bars). In addition, the transient FAK silencing abolished the reduction of fractional shortening, although it did not change the dilatation of the LVs in 12-week banded mice. Notably, no differences were seen in the systolic gradient across the aortic constriction between mice injected with siRNAGFP or siRNAFAK (supplemental Table II).
The average diameter of cardiac myocytes was reduced in banded mice treated with siRNAFAK in comparison with those treated with siRNAGFP (Figure 5A). In addition, FAK silencing was shown to attenuate the rise of the hypertrophic marker gene β-MHC induced by aortic banding (Figure 5B).
LV interstitial fibrosis of aortic-banded mice treated with siRNAFAK was attenuated, as compared with that of mice treated with siRNAGFP (Figure 5C through 5F). This was accompanied by attenuation of the increases in the LV collagen content induced by pressure overload, as indicated by the analysis of myocardial hydroxyproline in banded mice treated with siRNAFAK (Figure 5G). Moreover, FAK silencing prevented the increases in the zymogram gel lysis for MMP-2 induced by aortic banding. As shown in Figure 5H, LV samples from mice treated with siRNAGFP displayed increased gel lysis, whereas in samples from mice treated with siRNAFAK, this lysis was comparable to that from control mice treated with siRNAGFP or siRNAFAK. In fibroblasts harvested from 7-day banded mice, we confirmed the increases in FAK and MMP-2 expression induced by pressure overload and, additionally, we showed that FAK silencing canceled the rise in the expression of MMP-2 (Figure 5I).
siRNAFAK Reverses Established LV Hypertrophy
We next examined whether systemically administered siRNAFAK could reverse already-existing load-induced left ventricular hypertrophy. In these experiments, siRNAFAK or siRNAGFP were administered to 4-week aortic-banded mice with established hypertrophy but preserved left ventricular function. siRNAFAK reduced myocardial FAK expression (Figure 6A) to levels comparable to those of control mice (Figure 2). Typical structural and functional deterioration were seen in the LVs of banded mice treated with siRNAGFP (Figure 6B and supplemental Table III). In contrast, siRNAFAK reversed the left ventricular hypertrophy in banded mice. Noticeably, despite the regression of the hypertrophy, these mice had preserved left ventricular fractional shortening and less dilatation than those treated with siRNAGFP. These differences occurred despite the similarities in the increases of systolic aortic blood pressure and transconstriction gradient in both groups. In addition, siRNAFAK attenuated the increases in cardiac myocyte diameter and interstitial fibrosis in banded mice (supplemental Table III). Figure 6C and 6D shows representative examples of myocardial samples from banded mice treated with siRNAFAK or siRNAGFP. As shown in Figure 6E, survival was significantly higher in banded mice treated with siRNAFAK than in those treated with siRNAGFP.
The ability of systemically administered synthetic siRNA to efficiently reduce the amount of a specific target protein has been demonstrated in few disease models. In this study, we report the development of a strategy to target myocardial FAK by siRNA and demonstrate that transient myocardial FAK silencing can prevent, as well as reverse, mouse LV hypertrophy induced by aortic banding, despite the persistence of the pressure overload.
In Vivo FAK Silencing by siRNA
We demonstrated that a single intravenous injection of specific siRNA provided a prolonged reduction in FAK expression in both normal and hypertrophic mouse LVs. Importantly, we confirmed the silencing of FAK in cardiac myocytes and fibroblasts, thereby suggesting that myocyte and nonmyocyte myocardial cell populations may be equally susceptible to gene silencing by the RNA interference strategy reported here. These results indicate the feasibility of the use of RNA interference in the LVs, by using bolus injections via the jugular vein of naked siRNA, despite the expected low stability and poor cellular uptake of siRNA.18,20 Although such factors indeed limit the strategy of delivering siRNA systemically, it has been shown to be possible to circumvent such obstacles by administering comparatively large amounts of siRNA selectively into the organ where it needs to be delivered.19,21 Accordingly, we demonstrate herein that a modified strategy of local injection, ie, a bolus injection of a comparatively concentrated siRNA into the jugular vein, was effective in reducing FAK expression in the mouse LVs. The relatively modest FAK silencing in the kidney and the lack of silencing in the liver, as well as the demonstration that hydrodynamic injections of siRNAFAK via the tail vein did not change FAK expression in the mouse LVs further support the efficiency of the jugular delivery to induce in vivo gene silencing by naked siRNA. However, the relatively large amounts of siRNAFAK needed to obtain an efficient gene silencing might induce nonspecific and off-target effects and limit the use of this strategy. In this regard, a major nonspecific effect related to high concentrations of siRNA, observed as the interferon response,22 was shown to be absent in the mice of the present study. We also demonstrated that the treatment with siRNAFAK did not affect the expression of the FAK-related proline-rich tyrosine kinase 2 or the unrelated proteins extracellular signal-regulated kinase 1/2 or cardiac myosin, suggesting a lack of major off-target effects of this sequence. This is in agreement with recently reported data indicating that the majority of the off-target effects with systemic delivery of siRNA were attributable to the lipid-based transfection reagent and not to the siRNA.23
Remarkably, only at the third week after siRNAFAK injections did myocardial FAK transcript and protein expression recover to the levels seen in the LVs from mice that were treated with siRNAGFP. Although we did not expect such a prolonged effect, previous observations24,25 have indicated that gene silencing induced by naked siRNA may be effective for weeks. Such a prolonged effect has been attributed to the stability of the interaction of the active strand of siRNA with the RISC complex, which may persist for weeks, depending on the rate of cell division and the presence of the target mRNA within the cells.26,27 In this regard, it is worth mentioning that cardiac myocytes do not proliferate at a significant rate in normal or diseased hearts, although the other myocardial cell types may proliferate in response to pathophysiological stimuli. Interestingly, siRNAFAK reduced the myocardial FAK expression to comparable levels in the LVs of control and banded mice, indicating that the trophic influences of pressure overload did not affect the efficiency of siRNA in silencing myocardial FAK.
Effects of FAK Silencing in Chronically Overloaded LVs
A major finding of this study was that transient reductions of myocardial FAK expression by the RNA interference strategy markedly attenuated the development and reversed already-established left ventricular hypertrophy in aortic-banded mice. This implies that FAK is necessary not only to the development of but also to sustain LV hypertrophy in response to chronic pressure overload. The demonstration that reductions in FAK expression were paralleled by attenuation of the cardiac myocyte hypertrophy and expression of the hypertrophic gene marker β-MHC supports the conclusion that FAK silencing attenuates hypertrophic growth by affecting the responses of the cardiac myocytes to pressure overload. This is in line with previous data indicating that FAK is crucially involved in the hypertrophic response of cardiac myocytes to mechanical stress.6,7,14,15 However, influences arising in consequence of FAK silencing in the vessels of the peripheral circulation or neurohormonal factors might arguably contribute to the attenuation of the left ventricular hypertrophy in banded mice treated with siRNAFAK. Although we cannot exclude neither a significant FAK gene silencing in the systemic vasculature nor a reduction in the vascular resistance in banded mice treated with siRNAFAK, the demonstration here that mice treated with siRNAFAK or siRNAGFP had comparable transconstriction systolic gradients and increases in blood pressure in ascending aorta indicate that it is unlikely that reductions in the vascular resistance contributed substantially to the attenuation of the hypertrophic growth in mice treated with siRNAFAK. Further studies are necessary, however, to examine whether changes in coronary microcirculation or in the activity of the neurohormonal systems (eg, renin–angiotensin system) contribute to the antihypertrophic effect of myocardial FAK silencing.
Importantly, besides attenuating the hypertrophic growth, FAK silencing also mitigated the ongoing structural and functional deterioration of the hypertrophic LVs. Moreover, it has been shown that the abnormalities of chronically overloaded LVs were paralleled by enhanced myocardial FAK expression and activity. Thus, we reasoned that the persistently activated FAK signaling may be detrimental to hypertrophied LVs. This is apparently in conflict with the observations in myocyte restricted knockout mice14,15 that have indicated that depletion of FAK predisposes to a premature maladaptive remodeling of chronically overloaded LVs. We postulate that such contrasting effects might be related to the fact that myocardial FAK silencing, in contrast to the myocyte-restricted knockout, may attenuate the development of fibrosis by lowering FAK expression in fibroblasts, in addition to cardiac myocytes, favoring a better outcome of chronically overloaded LVs. The results of this study indeed support this hypothesis. Our results showed that the sites of myocardial fibrosis displayed remarkable anti-FAK staining, suggesting an activation of FAK signaling in the sites of fibrosis, as has been suggested previously in rat and human hypertrophied LVs.9,13 Accordingly, we demonstrated here that fibroblasts harvested from the LVs of 7-day banded mice had increased FAK expression. Moreover, it was shown here that the beneficial effects of FAK silencing in the hypertrophic remodeling of LVs was paralleled by a marked attenuation in the interstitial fibrosis, suggesting a role for FAK in the myocardial fibrogenesis induced by chronic pressure overload. Indeed, myocardial FAK silencing was shown to be accompanied by attenuation in the rises of myocardial collagen and MMP-2 activity, and, importantly, it was shown here that FAK silencing in fibroblasts harvested from overloaded myocardium was accompanied by a reduction in the MMP-2 expression, a molecule that has been shown to be a major determinant of the extracellular matrix remodeling process in overloaded myocardium.28,29
In summary, this study provides crucial information for the understanding of the importance of FAK signaling in the pathophysiology of cardiac hypertrophy and failure induced by chronic pressure overload. Furthermore, the demonstration that FAK silencing may prevent cardiac hypertrophy and its deterioration to maladaptive remodeling highlights the potential of FAK targeting by siRNA as an applicable therapeutic tool that may ameliorate remodeling and the outcome of cardiovascular diseases that evolve with hypertrophy and heart failure.
Sources of Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo grants 01/11698-1 and 04/10167-0 and Conselho Nacional de Desenvolvimento Científico e Tecnológico grant CNPq 521098/97-1.
Original received February 27, 2007; resubmission received July 30, 2007; revised resubmission received September 20, 2007; accepted October 4, 2007.
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