The contribution of mTOR signaling to cardiac hypertrophy that develops in response to a gradual increase in afterload, as occurs in the SHR, is unknown. In the present study we observed activation of mTOR signaling in hearts of young SHR during the developmental stage of cardiac hypertrophy. Pharmacologically inhibiting this pathway attenuated the extent of cardiac hypertrophy that ultimately occurs in this model. These are the first data indicating that mTOR signaling contributes importantly to cardiac hypertrophy in a clinically relevant model of hypertension.
Studies using pressure overloaded mice and guinea pigs reported that P70 S6 ribosomal kinase (S6K) phosphorylation is clearly correlated with cardiac hypertrophy21, 22
, and that cardiac-specific Akt overexpression increases activation of mTOR and results in cardiac hypertrophy9, 10, 23
. The in vivo
importance of mTOR has also been demonstrated in pressure overloaded rodents where rapamycin treatment results in inhibition of mTOR as determined by downstream effectors such as S6K, and attenuates cardiac hypertrophy evoked by aortic constriction21, 24, 25
. The studies using pressure-overloaded models are important, however, it should be noted that aortic constriction creates local hypertension, and does so in an abrupt manner. This process differs from the SHR that has gradually increasing systemic hypertension that eventually results in cardiac hypertrophy. The SHR may be considered to be more clinically relevant to the human experience where uncontrolled hypertension leads to cardiac hypertrophy. In the present study, rapamycin treated SHR demonstrated a clear and robust reduction in phosphorylation of S6 and 4E-BP1, both downstream targets of mTOR, and thus provided mechanistic evidence of the role of mTOR in the development of cardiac hypertrophy.
PKC isoforms have been reported to be mediators of cardiac function and hypertrophy. Overexpression or activation of PKCβII, ε and δ has been found to result in cardiac hypertrophy in mice26
. Interestingly, mice with deletion of PKCβ still develop cardiac hypertrophy in response to pressure overload or phenylephrine27
, and overexpression of PKCα does not cause cardiac hypertrophy, but results in diminished ventricular function 28
. Similarly, inhibition of conventional PKC isoforms (α, β, γ) increases cardiac function in mice29
, while adenoviral transfection of PKCα reduces cardiac contractility in the normally hypercontractile PKCα knockout mouse30
. In contrast to our original hypothesis, we did not find activation of any PKC isoform in 10 week-old SHR during the developmental phase of cardiac hypertrophy. However, 17 week-old SHR had an increase in p-PKCα/βII. While no other studies have examined PKC during the developmental phase of hypertrophy in the SHR, others have found PKCα, δ, ε activation in 6 month-old spontaneously hypertensive heart failure rats (SHHF)31
, and PKCβ activation in 16 week-old Dahl salt sensitive rats32
. Given the lack of PKC activation in hypertensive 10 week old SHR without hypertrophy, one may speculate that the PKC alterations in SHHF and Dahl salt sensitive rats with established hypertrophy may be related to regulation of cardiac function rather than growth.
In the present study, we found that treating SHR with rapamycin for 3 weeks resulted in even greater blood pressure compared to vehicle treated SHR. This is consistent with previous studies reporting detrimental changes in kidney function along with tubular atrophy and vascular pathology after treating with 0.8 mg/g rapamycin for 2 weeks 33, 34
. While hypertension has not been a reported side effect in human clinical trials using long term rapamycin treatment for immunosupression35
, it should be noted that clinical use employs a lower dose of rapamycin compared to animal studies such as the present one. Another observed side effect of rapamycin treatment was the weight loss that occurred in the SHR-Rap group. While no other studies have used rapamycin to attenuate hypertrophy in genetically hypertensive models, several studies have used similar dosages of rapamycin in pressure overloaded mice and rats. Studies using mice have not observed changes in body weight after either one 21, 25
or four weeks 20
of treatment with rapamycin, while pressure overloaded rats show significant weight loss even after just three days of rapamycin24
. To our knowledge the present study is the first to treat SHR with rapamycin.
It is seemingly contradictory that rapamycin treatment attenuated cardiac hypertrophy in SHR in spite of greater blood pressure, however, this underscores the importance of mTOR signaling in stimulating cardiac growth during the developmental phase of hypertrophy. Given the persistence of this pathological stimulus (hypertension) in SHR-Rap, it is also not surprising that expression of ANP and BNP, both markers of pathological cardiac hypertrophy or increased wall stress, remained increased. Therefore, it appears that the reduction in hypertrophy after rapamycin treatment is due solely to inhibition of mTOR, without fundamentally altering the pathological nature of the residual hypertrophy that develops in SHR-rap or the increase in wall stress. Given the disproportional degree of hypertrophy versus hypertension in rapamycin treated SHR, we used echocardiography to evaluate ejection fraction and fractional shortening as measures of cardiac function. We have previously reported that 16 week old SHR with cardiac hypertrophy have normal cardiac function compared to age matched WKY36
. In the present study, our data indicated that both ejection fraction and fractional shortening were also similar in vehicle and rapamycin treated SHR. We conclude that short-term rapamycin treatment does not adversely affect cardiac function, however, the effect of an extended (greater than 3 weeks) period of rapamycin treatment on cardiac function remains unknown. A limitation, however, of the present study is the lack of histological analysis of hearts from rapamycin treated and untreated SHR. Histological analyses from previous studies indicate that myocyte size is increased in 14 week-old SHR 37
while cardiac fibrosis develops between 12 to 20 months of age38
. However, in the present study, it is unknown to what degree rapamycin treatment may have altered cardiac structure with regard to myocyte size in 16 week-old SHR.
Recent studies have reported that sirolimus (rapamycin) treatment can reduce left ventricular (LV) hypertrophy in humans that is a side effect of kidney transplant39
and heart transplant40, 41
. Not surprisingly these reports have led to suggestions that rapamycin may have therapeutic potential for treating LV hypertrophy in cardiac transplant patients, as well as those with other etiologies such as hypertension and myocardial infarction. However, our data indicates several factors that should be seriously considered in this regard. For example, transplant patients examined in previous studies did not have hypertension40, 41
, or were under pharmacological blood pressure control39
, whereas SHR used in our study were severely hypertensive. This point is noteworthy since our data indicates that rapamycin in the face of untreated hypertension may worsen the condition. Second, although we achieved more substantial reductions in cardiac hypertrophy in our study (~50% reduction in developed hypertrophy) compared to those reported in the kidney or heart transplant patients (10% and ~5% reduction in LV mass index, respectively), our rapamycin doses were much higher in SHR vs. patients (2 mg/kg i.p. / day in SHR vs. human total dose of 1 mg p.o. / day). Though our data suggests that a larger reduction of hypertrophy may be possible in clinical situations, safety and efficacy studies would be required to determine a dose of rapamycin that would strike a balance between optimal reductions in hypertrophy and reduced mortality without severe side effects. Finally, we have demonstrated in the SHR model that rapamycin markedly inhibits S6 and 4E-BP1, however, it is unclear whether this near total inhibition is actually required to attenuate cardiac hypertrophy. As such, the possibility exists that lower doses of rapamycin might be similarly efficacious in this regard. In light of this, we believe that further research is warranted to elucidate the degree of mTOR inhibition that is associated with LVH regression before any assumptions can be made as to the potential therapeutic use of rapamycin in patients with hypertrophy due to other causes such as hypertension MI.