Ultrasonic Cavitation-Enabled Treatment for Therapy of Hypertrophic Cardiomyopathy: Proof of Principle

Abstract

Ultrasound myocardial cavitation-enabled treatment was applied to the SS-16^BN rat model of hypertrophic cardiomyopathy for proof of the principle underlying myocardial reduction therapy. A focused ultrasound transducer was targeted using 10-MHz imaging (10 S, GE Vivid 7) to the left ventricular wall of anesthetized rats in a warmed water bath. Pulse bursts of 4-MPa peak rarefactional pressure amplitude were intermittently triggered 1:8 heartbeats during a 10-min infusion of a microbubble suspension. Methylprednisolone was given to reduce initial inflammation, and Losartan was given to reduce fibrosis in the healing tissue. At 28 d post therapy, myocardial cavitation-enabled treatment significantly reduced the targeted wall thickness by 16.2% (p <0.01) relative to shams, with myocardial strain rate and endocardial displacement reduced by 34% and 29%, respectively, which are sufficient for therapeutic treatment. Premature electrocardiogram complexes and plasma troponin measurements were found to identify optimal and suboptimal treatment cohorts and would aid in achieving the desired impact. With clinical translation, myocardial cavitation-enabled treatment should fill the need for a new non-invasive hypertrophic cardiomyopathy therapy option.

Keywords: Cardiac fibrosis; Cardiac myocyte necrosis; Hypertrophic cardiomyopathy; Myocardial contrast echocardiography; Ultrasonic cavitation.

Figure 1

Photographs of a treated SS-16^BN rat heart from the acute and chronic studies. The light area in the heart (A) is the treated region with inflammation after 2 d. An H&E stained histological section (B) shows the wide region of inflammatory swelling (arrows). The blue color of the swollen region is due to numerous inflammatory cells with blue-stained nuclei. An optimally treated rat heart from the chronic study after 28 d. The light area in the heart (C) is the treated region with fibrous tissue appearing white. A Masson’s trichrome stained histological section (D) from the center of the treated region with fibrotic (scar) tissue at the treatment target (arrow) stained blue with Masson’s trichrome stain. The targeted region of the left ventricular wall is noticeably reduced in thickness. The scale bars are 5 mm.

Figure 2

Echocardiographic images of a sham-treated rat (top row) and a treated rat (bottom row) from the chronic study (8 sham and 14 treated rats). The time points were baseline (left column), 1 d (middle column) and 28 d (right column). The arrows in the left ventricle of the treated heart approximately indicate the treated region in the anterior myocardium, which had inflammatory swelling in a region larger than the beam path at 1 d and the narrower thickness reduction at 28 d.

Figure 3

Results for echocardiographic LV heat wall thickness from the chronic study. The echocardiographic results for the treated rats (blue symbols) n=14 was significantly increased above shams (black symbols) n=8 at day 1 and significantly reduced at day 28 (**, p<0.001). Standard error bars (green) include all data points. A suboptimal subset of the treated rats (red symbols) had no wall thinning.

Figure 4

Premature complex (PC) yield for the ultrasound pulse triggers (top) and the plasma troponin release (bottom) plotted as a function of the echocardiographic wall thinning. The PC data (n=19) has a sigmoidal trend (r²=0.83). The linear regression for the troponin results (r²=0.82) (n=19) shows proportionality to wall thinning. The low results for the suboptimal treatment (red symbols) recognized in Fig. 3 are clearly differentiated from the successful treatments (blue symbols) and shams (green symbols). Note that some shams have negative thinning (i. e., thickening) due to continued hypertrophy.

Figure 5

Analysis of echocardiography for the endocardial wall displacement (top) and the heart fractional strain in the treated region (bottom) plotted as in Fig. 3 with standard error bars in green. The treated wall displacement was significantly reduced (n= 11) after 1d when the tissue was somewhat swollen, and remained significantly reduced after 28d (*, p<0.05). Sham treated results were unchanged (n=8). A similar trend was even more distinct for wall strain (**, p<0.005). The sub optimal treatments (red symbols) had somewhat less impact relative to shams (black symbols) on these measures than did the optimal-treatments (blue symbols).

Figure 6

Histological images from a sham-treated rat heart (top row) and from an optimally treated heart (also shown in Fig. 1B) (Bottom row) from the chronic study (8 sham and 14 treated rats). Staining methods were hematoxylin and eosin (left column), Masson’s trichrome (middle column) and immunohistochemical staining with vimentin antibodies (right column). With hematoxylin and eosin, or Masson’s trichrome staining, many more cell nuclei (blue dots) are still evident in the fibrotic regions (blue with Masson’s trichrome). These cells are identified as numerous additional small fibroblasts and some endothelial cells in capillaries by the vimentin staining. The scale bar of 200 μm is the same for all images.