Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes

Figures

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  Figure 1. Shock wave treatment (SWT) prevents from heart failure after acute myocardial infarction. (A) Myocardial infarction was induced via LAD ligation. Subsequently, baseline measurement was preformed via echocardiography. Animals were treated with SWT immediately thereafter. Four weeks later, cardiac function was assessed via transthoracic echocardiography and pressure–volume measurement. (B) Baseline echo showed no difference between the groups before treatment. However, treated animals showed significantly improved left ventricular ejection fraction (LVEF) 4 weeks after treatment compared with untreated controls. (C) Representative M‐mode images from the transthoracic echocardiographies. The left ventricular anterior wall shows improved contractility after SWT compared to untreated controls. (D) Cardiac function was assessed in more detail using pressure–volume measurements. (E) Improved LVEF after SWT could be confirmed in pressure–volume measurements. In addition, treated animals showed decreased left ventricular end‐diastolic pressure (LVEDP; F), higher dP/dt max (G), decreased left ventricular end‐diastolic volume (LVEDV; H), dP/dt min. (I) and left ventricular end‐systolic…

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  Figure 2. Shock wave treatment (SWT) induces angiogenesis and avoids myocardial scar formation. (A) Representative images of myocardium analysed for capillaries (CD31) and arterioles (aSMA) via immunofluorescence staining. (B) SWT resulted in significantly increased numbers of capillaries in the myocardium. (C) In addition, animals showed an increased amount of arterioles after treatment. (D) Masson's trichrome staining was used to quantify the amount of fibrosis of the left ventricle. (E) We found a significantly decreased amount of fibrosis in treated animals. n = 6, *P < 0.05, ****P < 0.0001.

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  Figure 3. Shock wave treatment (SWT) causes RNA release and promotes cellular uptake. (A) Representative images of HUVECs treated with SWT and analysed for proliferation using Ki67 staining. (pink = Ki67, blue = DAPI, scale bar = 100 μm; B) SWT resulted in proliferation of HUVECs compared to untreated control cells. (C) Pre‐conditioned supernatant obtained from SW‐treated cells showed the same effect as direct SW application. (D) HUVECs were treated with SWT and analysed for RNA content (green = Syto16, scale bar = 100 μm). (E) We found significantly increased amounts of RNA in the supernatant of SW‐treated cells compared with untreated control supernatant. (F) Pre‐marked RNA was added to HUVECs prior to SWT. Subsequently, SWT was applied and RNA uptake quantified (red = poly(I:C), scale bar = 100 μm). (G) Cellular RNA uptake was significantly increased after SWT. Experiments were performed at least in triplicates and repeated at least twice. *P < 0.05, **P < 0.01.

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  Figure 4. RNA/protein complexes mediate SW effect. (A) SWT enhanced proliferation in HUVECs. The effect could not be abolished via RNase pre‐treatment, but only with additional proteinase treatment. (B) SWT resulted in increased segment formation, formation of junctions (C) and enhanced node formation (D) in a tube formation assay. Again, the effect could not be abolished by RNase treatment, but only with proteinase addition. (E) We found significantly more tubes after SWT of HUVECs. Experiments were performed at least in triplicates and repeated at least twice. *P < 0.05.

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  Figure 5. Increased LL37 expression after SWT. (A) Western blot analysis was performed to assess LL37 expression. (B) SW‐treated cells showed significantly increased LL37 expression after SWT compared to untreated controls. (C) Immunofluorescence staining for LL37 of HUVECs with and without treatment was performed. (D) After SWT, LL37 expression was significantly increased. (E) To assess LL37 release upon SWT, a LL37 ELISA of pre‐conditioned supernatants was performed. We found significantly increased amount of LL37 1 hr after SWT. (F) Finally, TLR3 activation after SWT was measured using a reporter cell assay. TLR3 activation was significantly increased after SWT. The effect was not abolished upon RNase addition, but only with addition of proteinase. Experiments were performed at least in triplicates and repeated at least twice. *P < 0.05.