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Published Online:ericyue.info/10.1096/fj.05-4232fje

SPECIFIC AIMS

Epidemiologic studies have shown that aging accounts significantly for the prevalence of erectile dysfunction (ED). The pathophysiology of ED during aging and its underlying molecular mechanisms are largely unknown. Evidence has shown that the RhoA/Rho-kinase signaling pathway maintains constriction of the cavernosal arterioles and sinuses, keeping the penis in the flaccid state. The aim of this study is to test the hypothesis that increased RhoA/Rho-kinase signaling pathway is a major factor in the pathogenesis of age-associated ED and the mechanism involves increased penile smooth muscle contractility through inhibition of myosin light chain phosphatase targeting subunit (MYPT1).

PRINCIPAL FINDINGS

1. Erectile function was impaired in aged rats

Erectile function was determined by measuring intracavernosal pressure (ICP), mean arterial pressure (MAP) and total ICP in young and aged rats upon electrical stimulation of the cavernous nerve. Erectile responses were significantly lower in aged rats at 2.5, 5, and 7.5 V (P<0.05). The ICP/MAP index was ∼50% less in aged rats at all voltages tested when compared with that in young rats. Similarly, total ICP (area under ICP curve) was also markedly reduced in aged rats after all three voltage stimulations (7.0±1.7, 10±2.4, and 10.7±1.7 at 2.5, 5 and 7.5 V for aged rats vs. 11.0±2.4, 17.8±2.5, and 20.4±3.0 at 2.5, 5, and 7.5 V for young rats, respectively). These data suggested that erectile responses were attenuated in aged rats.

2. Protein expressions of RhoA, Rho-kinase-α and -β, and MYPT1 were not altered while RhoA and Rho-kinase activities were increased in aged rat penes

RhoA, Rho-kinase-α and -β, and MYPT1 protein expressions were measured in young and aged rat penes using Western blot analysis. The protein levels were analyzed by densitometry and expressed as the ratio of RhoA, Rho-kinase, or MYPT1 per β-actin level. RhoA, Rho-kinase-α and -β, and MYPT1 protein expressions were similar in young and aged rat penes.

RhoA activity was determined by membrane-bound RhoA in young and aged penes. Our results show that membrane-bound RhoA was increased by 95 ± 15% in aged rat penes when compared with that in the young (P<0.05), indicating that RhoA activity was significantly increased in aged rat penes. To determine Rho-kinase activity, MYPT1 phosphorylation in young and aged penes was detected by a specific antibody recognizing MYPT1 phosphorylation at Thr696. Studies have shown that decreased myosin light chain (MLC) phosphatase activity is correlated with increased MYPT1 phosphorylation at Thr696. In this study, MYPT1 phosphorylation at Thr696 was significantly increased by 56 ± 8% in penile tissue isolated from aged rats when compared with that in young rats (P<0.05), suggesting an increase in Rho-kinase activity. This observation is confirmed by Rho-kinase activity assay results showing that aged rat penes have an approximately 2-fold greater Rho-kinase activity than young rat penes.

3. A Rho-kinase inhibitor improved erectile function in aged rats

To determine the effect of pharmacological inhibition of Rho-kinase on erectile function in young and aged rats in vivo, the selective Rho-kinase inhibitor, Y27632, was administered to the corpora cavernosa. Direct intracavernosal injection of Y27632 (10–100 nmol) caused increases in ICP/MAP and total ICP in both young and aged rats. However, there was a significantly greater increase in ICP/MAP and total ICP in response to Y27632 in aged rats at all doses studied, suggesting that Rho-kinase activity was increased in aged rat penises. The effect of Y27632 on erectile responses was dose-dependent. Injection 10, 30, and 100 nmol of Y27632 increased ICP/MAP to 0.46 ± 0.05, 0.58 ± 0.08, and 0.72 ± 0.05 in aged rats vs. 0.30 ± 0.04, 0.37 ± 0.06, and 0.47 ± 0.06 in young rats, respectively (P<0.05). Total ICP was raised to 81 ±17, 135 ± 26, and 192 ± 24 in aged rats at 10, 30, and 100 nmol of Y27632 vs. 30 ± 7, 40 ± 7, and 48 ± 11 in young rats (P<0.05). Local administration of Y27632 into the corpora cavernosa had no significant effect on MAP.

4. Dominant negative RhoA (T19NRhoA) gene transfer enhanced the erectile responses in aged rats

To confirm our observations that RhoA/Rho-kinase activity was elevated in aged rat penes, we targeted the RhoA/Rho-kinase signaling pathway at the molecular level via gene transfer of adeno-associated virus (AAV) encoding T19NRhoA to the corpora cavernosa of young and aged rats. Seven days after transfection with AAV carrying green fluorescence protein (AAVGFP), a reporter gene used as a vehicle control, or AAVT19NRhoA in young or aged rats, ICP and MAP were measured in response to electrical stimulation of the cavernous nerve. After AAVGFP transfer, ICP/MAP and total ICP were lower in aged rats when compared with that in young rats (P<0.05). These data are consistent with results obtained in rats without AAV treatment in which erectile function was comparatively reduced in aged animals, suggesting AAVGFP had no significant effect on erectile function. However, AAVT19NRhoA gene transfer to the corpora of young and aged rats significantly increased the ICP/MAP and total ICP to values greater than those observed in young and aged rats transfected with AAVGFP except at the 3 voltage setting (P<0.05, Fig. 1 A, B).

Figure 1.

Figure 1. Transfection of AAVT19NRhoA in penes of young and aged rats for 7 days improved erectile function in aged rats. A) ICP/MAP in response to cavernous nerve stimulation (CNS) in young rats transfected with AAVGFP (solid black bars) and young rats transfected with AAVT19NRhoA (blank bars). B) Total ICP in response to CNS in young rats transfected with AAVGFP and young rats transfected with AAVT19NRhoA. C). ICP/MAP in response to CNS in aged rats transfected with AAVGFP (solid black bars) and aged rats transfected with AAVT19NRhoA (blank bars). D) Total ICP in response to CNS in aged rats transfected with AAVGFP and aged rats transfected with AAVT19NRhoA. *P < 0.05, young vs. aged rats.

5. AAVT19NRhoA gene transfer suppressed the increased Rho-kinase activity in aged rat penes

After 7 days of AAVGFP treatment, Rho-kinase activity is 205 ± 29% higher in aged rat penes than that in young rat penes, which is similar to the difference between untreated aged and young rat penes. In AAVT19NRhoA-treated young rats, penile Rho-kinase activity is not significantly different from that in AAVGFP-treated young rats (P>0.05). However, gene transfer of AAVT19NRhoA to aged rat penes for 7 days significantly suppressed Rho-kinase activity to 117 ± 23% of that in AAVGFP-treated young rat penes (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

Normal erection is dependent on sufficient vasorelaxation induced by stimulation of the nervous system to overcome vasoconstriction of corporal smooth muscle. Reduced smooth muscle vasorelaxation or increased vasoconstriction leads to impaired erectile function. Evidence suggests that RhoA/Rho-kinase-mediated calcium sensitization plays a significant role in the regulation of corpora smooth muscle tone and maintains the penis in the flaccid state. Nevertheless, little information is available on the role of the RhoA/Rho-kinase signaling pathway in the process of aging in vasculature. It has been reported that RhoA mRNA expression and activity were increased in aortic and basilar arteries from aged rats relative to that from young rats, suggesting a possible role of RhoA in the development of vascular disease related to aging. A recent study showed that Y27632 administration improved erectile function in aged Brown-Norway rats, but the molecular mechanisms of age-associated ED induced by RhoA/Rho-kinase were not elucidated. A comparison of RhoA/Rho-kinase inhibition was not made between young and aged rats; therefore, it was not known whether increased RhoA/Rho-kinase activity is uniquely associated with aging.

We found that although protein expressions of these molecules in aged penes were not significantly different from those in young penes, the activities of RhoA and Rho-kinase were markedly increased, leading to the inactivation of MLC phosphatase and subsequently impaired erectile function. These data suggest that RhoA/Rho-kinase activity is regulated at post-translational levels in aged rats. Consistently, several studies have shown that increased RhoA/Rho-kinase activity is not completely dependent on RhoA, Rho-kinase, or MYPT1 protein expression levels. For example, in hypertensive rat models, RhoA or Rho-kinase protein expression in aorta was unchanged while MYPT1 phosphorylation level was significantly higher than that in normotensive rat aorta.

Increased RhoA/Rho-kinase signaling in aged rat penes was confirmed by the results of Rho-kinase activity assay showing that in aged rats Rho-kinase activity is 2-fold greater than that in young rats. Inhibition of Rho-kinase by Y27632 significantly improved erectile function in aged rats; effects on erectile function in young rats were less impressive. The limitation of using Y27632 to study the RhoA/Rho-kinase signaling pathway is that it has inhibitory effects on other kinases mediating smooth muscle contraction such as calcium-dependent protein kinase C (PKC) isoforms and MLC kinase. Recent studies have shown that Y27632 is a potent inhibitor of the calcium-independent PKC-δ and -ε isoforms, which increase calcium sensitivity through phosphorylation of a small protein called CPI-17, leading to inhibition of MLC phosphatase.

To distinguish RhoA/Rho-kinase-mediated calcium sensitization from PKC mediated calcium sensitization, we designed experiments by which AAVT19NRhoA was introduced to penes of young and aged rats to specifically target the RhoA/Rho-kinase pathway. Our results showed that gene transfer of T19NRhoA inhibited Rho-kinase activity and reversed ED in aged rats, providing convincing evidence that RhoA/Rho-kinase plays a significant role in inducing ED during the aging process. However, gene transfer of AAVGFP neither reduced Rho-kinase activity nor improved erectile function in aged rats, affirming the specific effect of T19NRhoA.

We did not investigate here how RhoA/Rho-kinase activity is increased in aged rat penes. It is likely that the increased RhoA/Rho-kinase activity in aged penes is due to decreased NO bioavailability. Studies have shown that NO inhibits RhoA activity through protein kinase G-dependent phosphorylation of RhoA and destabilizes GTP-RhoA membrane binding. In the penis, NO released from nerve endings or endothelial cells in penile tissue is the principal vasodilator. It has been reported that NO produced by neuronal NOS (nNOS) is decreased in aged rat penes because of decreased nerve endings. Calcium-independent endothelial NOS (eNOS) activity (phosphorylation at Ser1177) was significantly reduced, and phosphorylation of eNOS at Thr495, a negative regulatory site, was increased in aged Fischer 344 rats. Reduced endothelium-dependent smooth muscle relaxation associated with decreased eNOS protein expression and constitutive NOS activity in penile tissue was observed in Brown-Norway aged rats. Gene transfer of nNOS or eNOS to aged rat penes dramatically increased erectile responses to cavernous nerve electrical stimulation. Moreover, vascular aging is characterized by endothelial dysfunction due to excessive production of reactive oxygen species (ROS) and impaired redox defense systems including reduced superoxide dismutase activity. ED associated with increased levels of ROS was reported in aged rats, and erections were improved by gene transfer of extracellular superoxide dismutase to the aged rat penes. ROS may enhance RhoA/Rho-kinase activity through interacting with NO to form peroxynitrite, thereby decreasing NO bioavailability or by directly acting on the RhoA/Rho-kinase pathway.

Our hypothesis that increased RhoA/Rho-kinase activity is distinctively associated with ED during the aging process is supported by in vivo and in vitro experiments using pharmacologic and molecular tools (Fig. 2 ). To our knowledge, this is the first study to elucidate the role of RhoA/Rho-kinase in the pathogenesis of ED in aged rats at the molecular level. This work supports targeting the RhoA/Rho-kinase pathway for treatment of age-associated ED.

Figure 2.

Figure 2. Diagram of a proposed mechanism inducing age-associated ED. RhoA/Rho-kinase activity was abnormally elevated during the aging process. Rho-kinase inhibits myosin light chain phosphatase (MLCP) through phosphorylation of MYPT1, leading to an increase in myosin light chain (MLC) phosphorylation and corporal smooth muscle contraction. Increased corporal smooth muscle tone results in ED. Inhibition of the RhoA/Rho-kinase signaling pathway with T19NRhoA or Y27632 increases MLCP activity, restoring erectile function.

1These authors contributed equally to this work.

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