Preclinical model of type 1 diabetes and myocardial ischemia/reperfusion injury in conscious rabbits-demonstration of cardioprotection with rapamycin.

We developed a preclinical model of myocardial ischemia/reperfusion (I/R) injury in conscious diabetic rabbits to identify an early pharmacological intervention for patients with diabetes and acute myocardial infarction (AMI). Here, we describe a reproducible protocol for induction of diabetes with subsequent manifestation of myocardial I/R injury in conscious rabbits to mimic the real-life scenario observed in clinical settings. Further, we demonstrate the efficacy of rapamycin at the onset of reperfusion to limit the adverse effect of AMI. For complete details on the use and execution of this protocol, please refer to Samidurai et al. (2020).

Differential Regulation of mTOR Complexes with miR-302a Attenuates Myocardial Reperfusion Injury in Diabetes.

Persistent activation of mTOR (mammalian target of rapamycin) in diabetes increases the vulnerability of the heart to ischemia/reperfusion (I/R) injury. We show here that infusion of rapamycin (mTOR inhibitor) at reperfusion following ischemia reduced myocardial infarct size and apoptosis with restoration of cardiac function in type 1 diabetic rabbits. Likewise, treatment with rapamycin protected hyperglycemic human-pluripotent-stem-cells-derived cardiomyocytes (HG-hiPSC-CMs) following simulated ischemia (SI) and reoxygenation (RO). Phosphorylation of S6 (mTORC1 marker) was increased, whereas AKT phosphorylation (mTORC2 marker) and microRNA-302a were reduced with concomitant increase of its target, PTEN, following I/R injury in diabetic heart and HG-hiPSC-CMs. Rapamycin inhibited mTORC1 and PTEN, but augmented mTORC2 with restoration of miRNA-302a under diabetic conditions. Inhibition of miRNA-302a blocked mTORC2 and abolished rapamycin-induced protection against SI/RO injury in HG-hiPSC-CMs. We conclude that rapamycin attenuates reperfusion injury in diabetic heart through inhibition of PTEN and mTORC1 with restoration of miR-302a-mTORC2 signaling.

Reperfusion Therapy with Rapamycin Attenuates Myocardial Infarction through Activation of AKT and ERK.

Prompt coronary reperfusion is the gold standard for minimizing injury following acute myocardial infarction. Rapamycin, mammalian target of Rapamycin (mTOR) inhibitor, exerts preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R) injury. We hypothesized that Rapamycin, given at the onset of reperfusion, reduces myocardial infarct size through modulation of mTOR complexes. Adult C57 male mice were subjected to 30 min of myocardial ischemia followed by reperfusion for 1 hour/24 hours. Rapamycin (0.25 mg/kg) or DMSO (7.5%) was injected intracardially at the onset of reperfusion. Post-I/R survival (87%) and cardiac function (fractional shortening, FS: 28.63 ± 3.01%) were improved in Rapamycin-treated mice compared to DMSO (survival: 63%, FS: 17.4 ± 2.6%). Rapamycin caused significant reduction in myocardial infarct size (IS: 26.2 ± 2.2%) and apoptosis (2.87 ± 0.64%) as compared to DMSO-treated mice (IS: 47.0 ± 2.3%; apoptosis: 7.39 ± 0.81%). Rapamycin induced phosphorylation of AKT S473 (target of mTORC2) but abolished ribosomal protein S6 phosphorylation (target of mTORC1) after I/R. Rapamycin induced phosphorylation of ERK1/2 but inhibited p38 phosphorylation. Infarct-limiting effect of Rapamycin was abolished with ERK inhibitor, PD98059. Rapamycin also attenuated Bax and increased Bcl-2/Bax ratio. These results suggest that reperfusion therapy with Rapamycin protects the heart against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTORC1 and p38.

Long-acting PDE5 inhibitor tadalafil prevents early doxorubicin-induced left ventricle diastolic dysfunction in juvenile mice: potential role of cytoskeletal proteins.

The chemotherapeutic use of doxorubicin (Dox) is hindered due to the development of irreversible cardiotoxicity. Specifically, childhood cancer survivors are at greater risk of Dox-induced cardiovascular complications. Because of the potent cardioprotective effect of phosphodiesterase 5 (PDE5) inhibitors, we examined the effect of long-acting PDE5 inhibitor tadalafil (Tada) against Dox cardiotoxicity in juvenile mice. C57BL/6J mice (6 weeks old) were treated with Dox (20 mg/kg, i.v.) and (or) Tada (10 mg/kg daily for 14 days, p.o.). Cardiac function was assessed by echocardiography following 5 and 10 weeks after Dox treatment. The expression of cardiac proteins was examined by Western blot analysis. Dox treatment caused diastolic dysfunction in juvenile mice indicated by increasing the E/E' (early diastolic myocardial velocity to early tissue Doppler velocity) ratio as compared with control at both 5 and 10 weeks after Dox treatment. Co-treatment of Tada and Dox preserved left ventricular diastolic function with reduction of E/E'. Dox treatment decreased the expression of SERCA2 and desmin in the left ventricle; however, only desmin loss was prevented with Tada. Also, Dox treatment increased the expression of myosin heavy chain (MHCß), which was reduced by Tada. We propose that Tada could be a promising new therapy for improving cardiac function in survivors of childhood cancer.

Reperfusion therapy with recombinant human relaxin-2 (Serelaxin) attenuates myocardial infarct size and NLRP3 inflammasome following ischemia/reperfusion injury via eNOS-dependent mechanism.

AIMS: The preconditioning-like infarct-sparing and anti-inflammatory effects of the peptide hormone relaxin following ischemic injury have been studied in the heart. Whether reperfusion therapy with recombinant human relaxin-2, serelaxin, reduces myocardial infarct size and attenuates the subsequent NLRP3 inflammasome activation leading to further loss of functional myocardium following ischemia/reperfusion (I/R) injury is unknown. METHODS AND RESULTS: After baseline echocardiography, adult male wild-type C57BL or eNOS knockout mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min followed by 24 h reperfusion. Mice were treated with either serelaxin (10 µg/kg; sc) or saline 1 h prior to ischemia or 5 min before reperfusion. In both pre-treatment and reperfusion therapy arms, serelaxin improved survival at 24 h post MI in wild-type mice (79% and 82%) as compared with controls (46% and 50%, P = 0.01), whereas there was no difference in survival between serelaxin- and saline-treated eNOS knockout mice. Moreover, serelaxin significantly reduced infarct size (64% and 67% reduction, P < 0.05), measured with TTC staining, and preserved LV fractional shortening (FS) and end-systolic diameter (LVESD) in wild-type mice as compared with controls (P < 0.05). Interestingly, caspase-1 activity in the heart tissue, a measure of inflammasome formation, was markedly reduced in serelaxin-treated wild-type mice compared with controls at 24 h post-MI in both treatment modalities (P < 0.05). Genetic deletion of eNOS abolished the infarct-sparing and anti-inflammatory effects of serelaxin as well as functional preservation. Serelaxin plasma levels assessed at 5 min and 1 h after treatment, using ELISA, approximated physiologic relaxin levels during pregnancy in mice and parallels that in humans. CONCLUSION: Serelaxin attenuates myocardial I/R injury and the subsequent caspase-1 activation via eNOS-dependent mechanism.

Beet root juice protects against doxorubicin toxicity in cardiomyocytes while enhancing apoptosis in breast cancer cells.

Doxorubicin (DOX, Adriamycin) is a broad-spectrum chemotherapeutic drug used to treat a variety of cancers, although its clinical use is restricted by irreversible cardiotoxicity. Earlier studies show that beet root juice (BRJ), a natural and safe herbal product with high levels of nitrate and antioxidants, is a potent chemopreventive agent; however, its cardioprotective function is yet to be established. The goal of this study was to determine the protective effect of BRJ against DOX-induced cardiotoxicity, and its effect on DOX-induced cytotoxicity in MDA-MB-231 breast cancer cells. Adult rat cardiomyocytes and MDA-MB-231 cells were exposed to different concentrations of BRJ (0.5, 5, 50, 250, and 500 µg/ml) with or without DOX. Cell death, measured by trypan blue staining, was significantly reduced in cardiomyocytes but increased in MDA-MB-231 following 24 h of co-treatment with BRJ and DOX. Cell viability was also significantly reduced after BRJ and DOX co-treatment in MDA-MB-231 cells. Similarly, DOX-induced apoptosis, as determined by TUNEL assay, was significantly reduced following treatment with BRJ for 48 h in cardiomyocytes. In contrast, BRJ significantly increased DOX-mediated apoptosis in cancer cells with activation of poly(ADP-ribose) polymerase (PARP) and increased the Bax:Bcl-2 ratio. DOX-induced generation of reactive oxygen species (ROS) was reduced following co-treatment with BRJ in cardiomyocytes but increased dose-dependently with BRJ in MDA-MB-231 cells. In conclusion, lower concentrations of BRJ with DOX represented the most effective combination of cardioprotection and chemoprevention. These findings provide insight into the possible cardioprotective ability of BRJ in cancer patients treated with anthracycline chemotherapeutic drugs.

Inhibition of mammalian target of rapamycin protects against reperfusion injury in diabetic heart through STAT3 signaling.

Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.