Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell-Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium.

BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure

A New Therapeutic Modality for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin Induces Cardioprotection from Ischemia-Reperfusion Injury via Activation of PI3K/Akt Pathway and Anti-Inflammation in a Rat Model.

There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction (AMI), for which the effectiveness of interventional reperfusion therapy is hampered by myocardial ischemia-reperfusion (IR) injury. Pretreatment with statins before ischemia is shown to reduce MI size in animals. However, no benefit was found in animals and patients with AMI when administered at the time of reperfusion, suggesting insufficient drug targeting into the IR myocardium. Here we tested the hypothesis that nanoparticle-mediated targeting of pitavastatin protects the heart from IR injury.In a rat IR model, poly(lactic acid/glycolic acid) (PLGA) nanoparticle incorporating FITC accumulated in the IR myocardium through enhanced vascular permeability, and in CD11b-positive leukocytes in the IR myocardium and peripheral blood after intravenous treatment. Intravenous treatment with PLGA nanoparticle containing pitavastatin (Pitavastatin-NP, 1 mg/kg) at reperfusion reduced MI size after 24 hours and ameliorated left ventricular dysfunction 4-week after reperfusion; by contrast, pitavastatin alone (as high as 10 mg/kg) showed no therapeutic effects. The therapeutic effects of Pitavastatin-NP were blunted by a PI3K inhibitor wortmannin, but not by a mitochondrial permeability transition pore inhibitor cyclosporine A. Pitavastatin-NP induced phosphorylation of Akt and GSK3β, and inhibited inflammation and cardiomyocyte apoptosis in the IR myocardium.Nanoparticle-mediated targeting of pitavastatin induced cardioprotection from IR injury by activation of PI3K/Akt pathway and inhibition of inflammation and cardiomyocyte death in this model. This strategy can be developed as an innovative cardioprotective modality that may advance currently unsatisfactory reperfusion therapy for AMI

Effects of Pitavastatin-NP on cell death after IR.

<p><b>(A)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on MI size. N = 7 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(B)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on cytosolic cytochrome C in IR myocardium 30 minutes after reperfusion. N = 4 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on mitochondrial cytochrome C in IR myocardium 30 minutes after reperfusion. Data are mean±SEM (n = 4 per group). Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(D)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with ED-1 in AAR. Scale bar: 20 μm. <b>(E),</b> Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on ED-1-positive leukocyte (monocytes) infiltration in IR myocardium 24-hour after reperfusion. N = 7 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p

Effects of Pitavastatin-NP on RISK pathway.

<p><b>(A)</b>, Western blot analysis of phosphorylated Akt (Ser 473) in IR myocardium 3 hours after reperfusion. N = 6 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests. <b>(B)</b>, Western blot analysis of phosphorylated Akt in IR myocardium from animals treated with WM or with WM plus pitavastatin-NP, 3 hours after reperfusion. Data are mean±SEM (n = 6 per group) <b>(C)</b> Western blot analysis of phosphorylated Akt in IR myocardium from animals treated with FITC-NP or with pitavastatin-NP, 15 and 30 minutes after reperfusion. <b>(D)</b>, Representative photomicrographs of IR areas of hearts treated with FITC-NP (left) and Pitavastatin-NP (middle and right) stained immunohistochemically with antibody against phospho-Akt, and an expanded view of the boxed area of the middle panel (right). Scale bar: 100 μm. <b>(E)</b>, Western blot analysis of phosphorylated GSK3β (S9A) in IR myocardium 3 hours after reperfusion. N = 6 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p

Effects of Pitavastatin-NP on MI size.

<p><b>(A)</b>, Representative stereomicrographs of heart sections double-stained with Evans blue and TTC 24 hours after reperfusion. Scale bar: 5 mm. <b>(B),</b> Effects of Pitavastatin-NP and pitavastatin alone on MI size at the time of reperfusion. N = 6–10 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Quantification of Area at risk in the group treated with pitavastatin-NP or pitavastatin alone. Data are mean are (n = 6–10 per group) Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests.</p

Experimental protocols.

<p>Adult male Sprague-Dawley (SD) rats, 8 weeks of age were used. Experimental protocol 1: At the time of reperfusion, animals were divided into 3 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC alone (FITC 0.33 mg in saline 3.3 mL/kg), or 3) FITC-NP (PLGA 8.3 mg containing 0.33 mg FITC in saline 3.3 mL/kg). Three hours after reperfusion, animals were sacrificed. The left lower panel shows representative stereomicrographs of heart sections double-stained with Evans blue and TTC: the MI area (TTC negative, white), non-MI area within AAR (TTC positive/Evans blue negative, red), non-ischemic area (TTC positive/Evans blue positive, purple) and AAR (Evans blue negative). Experimental protocol 2: At the time of reperfusion, animals were divided into 4 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC-NP (PLGA 8.3 mg/kg in saline 3.3 mL/kg), 3) pitavastatin (1.0 and 10 mg/kg in saline 3.3 mL/kg), or 4) pitavastatin-NP (PLGA containing of 0.1 and 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 3: Animals were divided into 3 groups receiving administration of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) vehicle (saline 3.3 mL/kg) after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia, 3) pitavastatin-NP (PLGA containing of 1.0 mg/kg pitavastatin in saline 3.3 mL/kg) after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia. Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 4: To examine the effects of Pitavastatin-NP on left ventricular function after IR, animals were divided into 3 groups that received intravenous injection of the following drugs at the time of reperfusion: 1) vehicle (saline 3.3 mL/kg), 2) pitavastatin alone (1.0 mg/kg in saline 3.3 mL/kg) or 3) Pitavastatin-NP (PLGA containing 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Echocardiography and measurement of systolic blood pressure and heart rate by using tail-cuff method were performed at baseline and 2-day, 1-week, 2-weeks and 4-weeks after reperfusion.</p

Effects of Pitavastatin-NP on cell death after IR.

<p><b>(A)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on MI size. N = 7 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(B)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on cytosolic cytochrome C in IR myocardium 30 minutes after reperfusion. N = 4 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on mitochondrial cytochrome C in IR myocardium 30 minutes after reperfusion. Data are mean±SEM (n = 4 per group). Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(D)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with ED-1 in AAR. Scale bar: 20 μm. <b>(E),</b> Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on ED-1-positive leukocyte (monocytes) infiltration in IR myocardium 24-hour after reperfusion. N = 7 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p

Leukocyte counts and Plasma biomarker profile 24 hours after IR.

<p>Data are expressed as the mean ± SEM (N = 6 each).</p><p>*<i>P</i><0.05 versus vehicle group. IR: ischemia-reperfusion.</p><p>Leukocyte counts and Plasma biomarker profile 24 hours after IR.</p

Effects of Pitavastatin-NP on inflammation and apoptosis in IR myocardium 24 hours after reperfusion.

<p><b>(A)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with NF-B (p65 subunit), MCP-1, ED-1 and TUNEL. Scale bar: 20 μm. <b>(B)</b>, Quantification of the number of NF-B (p65 subunit) positive cells, the MCP-1-positive area, ED-1-positive leukocytes (monocytes) in IR myocardium and the number of TUNEL-positive cells in infarct border myocardium 24 hours after reperfusion. Data are mean±SEM (n = 5–8 per group). Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p