Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Acute canagliflozin treatment protects against in vivo myocardial ischemia-reperfusion injury in non-diabetic male rats and enhances endothelium-dependent vasorelaxation.

BACKGROUND: The sodium-glucose cotransporter-2 (SGLT2) inhibitor canagliflozin has been shown to reduce major cardiovascular events in type 2 diabetic patients, with a pronounced decrease in hospitalization for heart failure (HF) especially in those with HF at baseline. These might indicate a potent direct cardioprotective effect, which is currently incompletely understood. We sought to characterize the cardiovascular effects of acute canagliflozin treatment in healthy and infarcted rat hearts. METHODS: Non-diabetic male rats were subjected to sham operation or coronary artery occlusion for 30 min, followed by 120 min reperfusion in vivo. Vehicle or canagliflozin (3 µg/kg bodyweight) was administered as an intravenous bolus 5 min after the onset of ischemia. Rats underwent either infarct size determination with serum troponin-T measurement, or functional assessment using left ventricular (LV) pressure-volume analysis. Protein, mRNA expressions, and 4-hydroxynonenal (HNE) content of myocardial samples from sham-operated and infarcted rats were investigated. In vitro organ bath experiments with aortic rings from healthy rats were performed to characterize a possible effect of canagliflozin on vascular function. RESULTS: Acute treatment with canagliflozin significantly reduced myocardial infarct size compared to vehicle (42.5 ± 2.9% vs. 59.3 ± 4.2%, P = 0.006), as well as serum troponin-T levels. Canagliflozin therapy alleviated LV systolic and diastolic dysfunction following myocardial ischemia-reperfusion injury (IRI), and preserved LV mechanoenergetics. Western blot analysis revealed an increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric-oxide synthase (eNOS), which were not disease-specific effects. Canagliflozin elevated the phosphorylation of Akt only in infarcted hearts. Furthermore, canagliflozin reduced the expression of apoptotic markers (Bax/Bcl-2 ratio) and that of genes related to myocardial nitro-oxidative stress. In addition, treated hearts showed significantly lower HNE positivity. Organ bath experiments with aortic rings revealed that preincubation with canagliflozin significantly enhanced endothelium-dependent vasodilation in vitro, which might explain the slight LV afterload reducing effect of canagliflozin in healthy rats in vivo. CONCLUSIONS: Acute intravenous administration of canagliflozin after the onset of ischemia protects against myocardial IRI. The medication enhances endothelium dependent vasodilation independently of antidiabetic action. These findings might further contribute to our understanding of the cardiovascular protective effects of canagliflozin reported in clinical trials.