Experimental protocol.

<p>The instrumentive surgery of the left anterior descending coronary artery (LAD-Instrumentation) was performed seven days prior to the acute myocardial infarction and reperfusion (AMI/R) to avoid a pro-inflammatory influence of trauma on post-infarction remodelling (PIR). The treatment group received UMS four days after AMI/R. On day +4 and +14, a reconstructive 3-dimensional echocardiography (r3DE) was performed to quantify global and regional left-ventricular function. Additionally, r3DE was carried out with low-dose dobutamine on day +14. Ultimately, hearts were harvested for histological workup.</p

RNA-levels after UMS.

<p>UMS increases VEGF-a, IGF-1 and Cav-3 mRNA-levels within 15 min and reaches its peak expression after 6 hours. A prolonged upregulation could not be observed longer than 30 hours after UMS as compared to controls. The displayed p-values refer to the comparison with sham-treated animals.</p

Histological analyses.

<p>Myocardial scar formation was determined by means of histomorphometry. (A) Representative images of short axis mouse heart sections stained with picrosirius-red and fast-green without (left) and with (right) UMS-treatment. The collagenous tissue is coloured red whereas myocardium is green. (B) UMS-treated mice demonstrated a significantly lower collagenous scar burden compared to control mice.</p

Global and regional left-ventricular function.

<p>(A) Mean left-ventricular ejection fraction (LV-EF) was moderately reduced four days after acute myocardial infarction and reperfusion (AMI/R) prior to ultrasound-mediated stimulation of microbubbles (UMS) in both groups. (B) Regional LV-function was quantified by means of reconstructive 3-dimensional echocardiography (r3DE) and is expressed as fraction of akinetic myocardial mass. UMS-treated animals (circles) demonstrated functional improvement two weeks after AMI/R, as compared to controls (squares). (C, D) Global and regional LV-function were obtained prior and during pharmacological stimulation with low-dose dobutamine on day +14. Inotropic response was preserved in both groups and revealed a significant increase in LV-EF and decrease in fraction of akinetic myocardial mass. Both non-invasive measures are parameters indicating preserved myocardial viability after AMI and reperfusion. In all, UMS improved LV-function after AMIR/R without impact on myocardial viability.</p

Microvascular density assessed by CD31 staining.

<p>(A) Microvascular density was assessed in the myocardial scar, both adjacent borderzones, and non-infarcted regions (posterior left-ventricular wall). No differences between untreated and UMS-treated animals were found with respect to the scar tissue and the non-infarcted regions. However, the myocardial borderzone tissue of UMS-treated mice revealed a significantly increased microvascular density as compared to non-treated animals. (B) Representative CD31 stained histological images of scar, untreated borderzone (−UMS), and UMS-treated borderzone (+UMS) (from left to right). CD31 positive vessels can be identified by their dark colour. Note the higher microvascular density of the UMS-treated mice compared to the non-treated group. In contrast, scar displayed the lowest microvascular density.</p

Schematic illustration for determination of hypertrophy.

<p>The illustration shows the six locations of wall thickness measurements in a mid-ventricular histological short axis section with scarring of the anterolateral left-ventricular wall. S: scar; AS: anteroseptal borderzone; AL: anterolateral borderzone. Control regions: IS: inferoseptal wall; I: inferior wall; IL: inferolateral wall.</p

Myocardial protein concentration of IGF-1 after UMS.

<p>Insulin-like growth factor 1 (IGF-1) was measured with a quantitative ELISA. UMS application not only increased IGF-1 content in control hearts, but also demonstrated a significant upregulation of IGF-1 on top of acute myocardial infarction and reperfusion (AMI/R).</p

Delivery of myocardial UMS in mice.

<p>UMS-mediated myocardial delivery of fluorescent nanospheres was measured and quantified by fluorescence microscopy. Myocardial delivery of fluorescent nanospheres with UMS was feasible in mice and demonstrated a dose-dependent effect.</p