Specific inhibition of mTORC1 improves cardiac function and reduces infarct size after ischemia/reperfusion

Myocardial infarction (MI) leads to irreversible cell death in the infarct area and to remodeling of viable myocardium in the border area due to the ischemic stress, both of which lead to scarring in the form of fibrosis. Previous studies showed that transient inhibition of the mTOR pathway can attenuate pathological remodeling of the myocardium and improve cardiac function but is associated with significant systemic off-target effects that limit clinical applicability.

The mTOR kinase forms two different complexes in cardiomyocytes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is predominantly involved in the regulation of cell growth and proliferation, whereas mTORC2 is accountable for cell survival.

We used a novel mTORC1-selective inhibitor, which outperforms previously clinically used mTOR inhibitors such as Rapamycin or Everolimus in its potency and selectivity for mTORC1 over mTORC2. Since inhibition of mTORC1 and thus a shift towards protective mTORC2 signaling leads to a reduction of apoptosis in cardiomyocytes, we wanted to determine whether these properties make specific mTORC1 inhibitors more suitable for reducing infarct size than other mTOR inhibitors.

 

Efficacy, dosing and mTORC1-selectivity of mTORC1 specific inhibitors were tesed in mice, confirming the safety and high selectivity in the heart for mTORC1 over MTORC2. Multiple in vitro experiments showed its safety and baseline effects. Next, we investigated cardiac function and infarct size after ischemia/reperfusion surgery in mice and the baseline effects in vivo and in vitro.

We used an ultrasound-guided, minimally invasive approach to induce MI in mice (45min ischemia time after which reperfusion was performed). Inhibitors were administered on day 0, 1, 2 and 7 post-infarction or as a vehicle. Cardiac function was assessed at baseline and weekly post-infarction by B-mode imaging. Infarct size was measured by Masson's trichrome staining and Axioscan analysis. qPCR analyses were performed for fibrosis and heart failure markers. Immunophenotyping of cardiac lysates 48 hours after infarction was measured by FACS analysis to visualize the extent of inflammation after MI.

After ischemia/reperfusion, the mTORC1 inhibition caused improved cardiac function than the control group. Histologic staining of the left ventricle and reduction of Col1A and NT-proBNP detected by qPCR analysis showed a decrease in infarct size after mTORC1 inhibition. The inflammatory response determined by FACS analysis was reduced with regard to the migration of leukocytes (neutrophils, monocytes/macrophages) into the tissue after mTORC1 inhibition. FACS apoptosis assay showed the safety on neonatal rat cardiomyocytes (NRCMs) and cardiomyocyte contractility assessed with the IonOptix CytoCypher MultiCell Throughput System showed no change in isolated adult rat cardiomyocytes under normal conditions and upon adrenergic stimulation with isoprenaline.

In addition, anti-remodeling properties of mTORC1 inhibitors were demonstrated as it was shown to be effective in preventing phenylephrine-induced hypertrophy in NRCMs as measured by the IN Cell Analyzer. 

 

Selective mTORC1 inhibition results in reduced infarct size in mice after ischemia/reperfusion, improves cardiac function and reduced cardiac inflammation.