Delivery of Hydrogen Sulfide by Ultrasound Targeted Microbubble Destruction Attenuates Myocardial Ischemia-reperfusion Injury

Abstract

Hydrogen sulfide (H2S) is an attractive agent for myocardial ischemia-reperfusion injury, however, systemic delivery of H2S may cause unwanted side effects. Ultrasound targeted microbubble destruction has become a promising tool for organ specific delivery of bioactive substance. We hypothesized that delivery of H2S by ultrasound targeted microbubble destruction attenuates myocardial ischemia-reperfusion injury and could avoid unwanted side effects. We prepared microbubbles carrying hydrogen sulfide (hs-MB) with different H2S/C3F8 ratios (4/0, 3/1, 2/2, 1/3, 0/4) and determined the optimal ratio. Release of H2S triggered by ultrasound was investigated. The cardioprotective effect of ultrasound targeted hs-MB destruction was investigated in a rodent model of myocardial ischemia-reperfusion injury. The H2S/C3F8 ratio of 2/2 was found to be an optimal ratio to prepare stable hs-MB with higher H2S loading capability. Ultrasound targeted hs-MB destruction triggered H2S release and increased the concentration of H2S in the myocardium and lung. Ultrasound targeted hs-MB destruction limited myocardial infarct size, preserved left ventricular function and had no influence on haemodynamics and respiratory. This cardioprotective effect was associated with alleviation of apoptosis and oxidative stress. Delivery of H2S to the myocardium by ultrasound targeted hs-MB destruction attenuates myocardial ischemia-reperfusion injury and may avoid unwanted side effects.

Figure 1. Characterization of hs-MBs prepared with different H₂S/C₃F₈ ratios.

(A) Appearance of hs-MBs prepared with different H₂S/C₃F₈ ratios. (B) hs-MBs under optical microscope. (C) Concentration of hs-MBs. (D) Mean diameter of hs-MBs. (E) Amount of H₂S encapsulated in hs-MBs. (F) Stability of hs-MBs. (G) Size distribution of hs-MB prepared with the H₂S/C₃F₈ ratio of 2/2. *P < 0.01, vs H₂S/C₃F₈ ratio of 4/0; ^#P < 0.01, vs H₂S/C₃F₈ ratio of 3/1; ^δP < 0.05, vs baseline (at 0 h). hs-MB indicates microbubble loaded with hydrogen sulfide; H₂S/C₃F₈, volume ratio of hydrogen sulfide and octafluoropropane.

Figure 2. Ultrasound triggered H₂S release from hs-MB in vitro.

(A) In vitro setup of flow system for ultrasound triggered H₂S release from hs-MB. (B) Baseline level of H₂S. (C) Change of H₂S level during hs-MB infusion. (D) Change of H₂S level during hs-MB infusion and ultrasound irradiation. (E) Comparison of maximum concentration of H₂S. *P < 0.05, vs hs-MB. US indicated ultrasound; hs-MB, microbubble loaded with hydrogen sulfide.

Figure 3. In vivo local delivery of H₂S mediated by hs-MB and US.

(A) In vivo imaging of ultrasound targeted hs-MB destruction in the myocardium. Red dotted line indicated the region of myocardium. White arrow indicated the ultrasound pulse emitted from an ultrasonic cavitation apparatus. (B) Comparison of H₂S concentration in various tissues following treatment. *P < 0.05 vs Control. US indicated ultrasound; hs-MB, microbubble loaded with hydrogen sulfide.

Figure 4. Ultrasound targeted hs-MB destruction limit the extent of MIR.

(A) In vivo experimental protocol showing the groups, intervention and outcomes measurement. (B) Electrocardiogram of rats underwent myocardial ischemia and reperfusion. (C) Ultrasound imaging of myocardial perfusion during ischemia or reperfusion. Red dotted line indicated the region of myocardium. Red arrows indicated perfusion defect in the anterior wall. (D) Representative photographs of mid-ventricular cross sections stained with Evans/TTC. Dark blue stain indicated viable area; White stain indicated infarct region; White plus red stain indicated area at risk. (E) Comparison of area at risk per left ventricle (AAR/LV). (F) Comparison of area of infarct size normalized to the area at risk (IS/AAR). *P < 0.05. MIR indicates myocardial ischemia-reperfusion injury; c-MB, control microbubble; US, ultrasound; hs-MB, microbubble loaded with hydrogen sulfide; NS, not significant.

Figure 5. Ultrasound targeted hs-MB destruction preserved left ventricular function.

(A) The representative echocardiograph images of short-axis view of the left ventricle in M-mode. (B) Left ventricular end- diastolic diameter (EDd); (C) Left ventricular end-systolic diameter (ESd); (D) Left ventricular fractional shortening (LVFS); (E) Left ventricular ejection fraction (LVEF). *P < 0.05. MIR indicates myocardial ischemia-reperfusion injury; c-MB, control microbubble; US, ultrasound; hs-MB, microbubble loaded with hydrogen sulfide; NS, not significant.

Figure 6. Ultrasound targeted hs-MB destruction alleviated MIR induce apoptosis and oxidative stress.

(A) Representative pictures of sections stained with TUNEL (×400). Green fluorescence indicated TUNEL-positive apoptotic nuclei; blue fluorescence indicated total cardiomyocyte nuclei. (B) Quantification of the TUNEL positive cell. (C) MDA level in the myocardium. (D) SOD level in the myocardium. *P < 0.05; ^#P < 0.01. MIR indicates myocardial ischemia-reperfusion injury; c-MB, control microbubble; US, ultrasound; hs-MB, microbubble loaded with hydrogen sulfide; MDA, malondialdehyde; SOD, superoxide dismutase; NS, not significant.