Selective mTORC1 inhibition attenuates myofibroblast activation and improves cardiac function in response to pressure overload

Introduction:
In response to chronic pressure overload cardiac fibroblasts (CF) become activated and differentiate into myofibroblasts. Recent studies suggest that targeting cardiac fibrotic remodeling may be beneficial, but effective anti-fibrotic treatments have not yet been established in clinical practice. The protein kinase complex mTORC1 is a central regulator of cellular function and mTORC1 is known to represent a critical signaling node of activated fibroblasts during organ fibrogenesis. However, the function of mTORC1 in cardiac fibrosis and whether it can be selectively targeted to attenuate fibrosis has not been investigated.

Methods:

Primary neonatal rat CFs were studied in vitro. To induce chronic pressure overload, male 10 weeks old C57BL/6N mice were operated by o-ring-aortic banding (ORAB). In addition to the classical mTOR inhibitors Rapamycin and Torin1, we investigated a novel mTORC1-selective compound (C1-inhib) to improve complex specificity. mTORC1 was also genetically inhibited using siRNA targeting the mTORC1 component RAPTOR. Myofibroblast activity was examined using a collagen gel contraction assay. In vivo, cardiac function was assessed by echocardiography. Fibrosis was assessed by Sirius Red staining.

Results:

Compared to Rapamycin or Torin1, C1-inhib selectively inhibited the phosphorylation of all examined mTORC1 targets. While stimulation with TGF-β resulted in strong contraction of vehicle treated CF-embedded collagen gels, Rapamycin, Torin1 or C1-inhib all significantly reduced gel contraction. Similar findings were observed by genetic mTORC1 inhibition. In vivo, ORAB surgery resulted in cardiac hypertrophy and fibrosis. Postoperatively mice were treated with C1-inhib on days 1, 3, and weekly thereafter, a protocol that was found effective in preclinical rodent cancer trials.

Additionally, a set of mice received first treatment seven days after surgery, allowing hypertrophy to develop before treatment initiation. Echocardiograms were performed weekly, and after 4 weeks, hearts were collected for analysis. Mice treated with C1-inhib showed a trend for attenuated hypertrophy but did not reach significance. Cardiac contractility was improved with C1-inhib treatment both with immediate treatment and notably, even when treated seven days after surgery.

Conclusions:
In summary, this study demonstrates the involvement of mTORC1 in the activation and contractile response of cardiac myofibroblast. Using C1-inhib we demonstrate for the first time the feasibility of pharmacological full mTORC1 inhibition in cardiac cells and its optimized pharmacological properties compared to classical mTOR inhibitors. In vivo, C1-inhib resulted in improved cardiac function in response to pressure overload, both with early and late treatment initiation. In conclusion, we show that pharmacological mTORC1-selective inhibition is feasible bypassing clinical limitations of current mTOR inhibitors.