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Online Appendix for the following JACC article
TITLE: Real-Time Assessment of Myocardial Contractility Using Shear Wave Imaging
AUTHORS: Mathieu Pernot, PhD, Mathieu Couade, MSc, Philippe Mateo, PhD, Bertrand
Crozatier, MD, PhD, Rodolphe Fischmeister, PhD, Mickaël Tanter, PhD
Supplemental Methods
Ex vivo physiology. Adult male Wistar rats (521±109g, n=6) were anesthetized by
intraperitoneal injection of pentobarbital (150 mg/kg). The heart was quickly removed and
placed in oxygenated Krebs-Henseleit solution (95% O2 and 5 % CO2 , pH 7.35) containing (in
mmol/L): NaCl 113, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 from 0.8 to 2.5,
glucose 11, and mannitol 1.1. The aorta was cannulated and perfused by the Langendorff method
with oxygenated Krebs-Henseleit solution at constant pressure (75 mmHg, temperature
37±0.2°C). A latex water-filled balloon was inserted into the LV chamber and connected to a
pressure transducer (Statham gauge Ohmeda, Bilthoven, Holland) for continuous measurement
of isovolumic heart pressure. Heart rate, LV developed pressure (LVDP) and the first derivatives
of LV pressure (LV +dP/dtmax and LV -dP/dtmax) were measured online using dedicated
software (Emka, Paris, France). The isolated hearts were immersed into a saline bath (50x50x50
mm3) and the ultrasonic array was positioned through an acoustic window performed on the side
of the water tank (Supplemental Figure 1).
Induction of cardiac hypertrophy in rats. Adult male Wistar rats (520±46g) were also used in
this study. Left ventricular hypertrophy was induced at three weeks of age (body weight <60 g)
according to previously described methods (1). The animals were anesthetized (pentobarbital 60
mg/kg), the thorax was opened and a stainless steel hemoclip of 0.6 mm ID was placed on the
ascending aorta. Age-matched control animals (sham-operated) underwent the same procedure
without placement of the clip. Studies were performed 4 months after the surgical procedure.
Ultrafast imaging. The propagation of the shear wave was imaged at a very high frame rate
(12,000 frames/s). This was done by transmitting a single full-aperture and quasi-plane ultrasonic
wave transmit. With only one firing, the frame rate equals the pulse repetition frequency (PRF)
than can be as high as 20,000 frames per second depending on the imaging depth and the
transducer aperture. In our case, the frame rate was fixed at 12,000 Hz. Experimentally, an
acquisition of the shear wave propagation can be performed in less than 5 ms. Thus, the local and
quantitative stiffness of the cardiac muscle could theoretically be performed up to 200 times per
second. Nevertheless, due to technical limitations such as probe heating, safety considerations
(acoustic power limitation for regulatory issues) and memory size on the ultrasound acquisition
boards, the actual maximal elasticity frame rate is below this theoretical maximal frame rate. As
illustrated in Supplemental Fig. 2, the elastography sequence is here repeated up to 40 times with
a fixed and constant repetition time between two consecutive acquisitions (7.5 ms). The shear
wave propagation is imaged during the first 5 ms following the shear wave generation (60
frames). The repetition time is adjusted with respect to the heart rate: the complete sequence
covers at least a full cardiac cycle. In addition, a trigger signal is generated by the ultrasound
scanner at the beginning of each pushing beam. This signal is recorded simultaneously to the
ECG and LV pressure using a dedicated acquisition board with multiple synchronous acquisition
channels (g.USBamp, gtec, Austria).
Supplemental Results
Heart rate dependence of myocardial stiffness. Supplemental Figure 3 shows heart rate
dependence of myocardial stiffness. The systolic shear modulus was found to decrease slightly
with increasing cardiac contraction rate (approximately 30% decrease when the heart rate almost
doubled). The decreased contractility with increased pacing rate was not surprising since, unlike
in higher mammals, the negative force-frequency relationship is a characteristic of rat heart (3).
In contrast, diastolic stiffness was found to increase with heart rate and almost doubled when
heart rate increased from 300 to 560 bpm. This effect could be explained by an incomplete
relaxation of the myocardium at high heart rate when the time constant of isovolumic pressure
fall is longer than the duration of diastole (4).
Myocardial stiffness during β-adrenergic stimulation in LV hypertrophy model. The
inotropic effect of a β-adrenergic stimulation on myocardial stiffness was investigated by
perfusing isoproterenol in normal and hypertrophic hearts. Baseline measurements performed on
the hearts at rest are shown in Supplemental Table 1. The myocardium was found significantly
stiffer in hypertrophied hearts than in sham operated in diastole. This could be explained by
myocardial fibrosis commonly associated with hypertrophy that tends to stiffen the myocardium
(2). Supplemental Figure 4 shows the myocardial systolic stiffness response for sham and
stenosed hearts at different concentrations of isoproterenol (10-9, 10-8 and 10-7 M). In normal
hearts, systolic stiffness was found to increase strongly upon administration of isoproterenol (up
to 22.9±4.5 kPa) whereas no significant change was found in hypertrophic hearts. Diastolic
stiffness was unchanged in all hearts (not shown).
References
1. Abi-Gerges A, Richter W, Lefebvre F, Mateo P, Varin A, Heymes C, Samuel J, Lugnier C,
Conti M, Fischmeister R, Vandecasteele G. Decreased expression and activity of cAMP
phosphodiesterases in cardiac hypertrophy and its impact on β-adrenergic cAMP signals.
Circ Res 2009;105:784-792.
2. Conrad CH, Brooks WW, Hayes JA, Sen S, Robinson KG, Bing OHL. Myocardial fibrosis
and stiffness with hypertrophy and heart failure in the spontaneously hypertensive rat.
Circulation 1995;91:161-170.
3. Endoh M. Force-frequency relationship in intact mammalian ventricular myocardium:
physiological and pathophysiological relevance. Eur J Pharmacol 2004;500:73-86.
4. Weisfeldt ML, Frederiksen JW, Yin FCP, Weiss JL. Evidence of incomplete left ventricular
relaxation in the dog. J Clin Invest 1978;62:1296-1302.
Supplemental Figure 1: Experimental setup with Langendorff perfused rat heart.
Supplemental Figure 2: Complete Shear wave imaging acquisition sequence for dynamic and
real time imaging of cardiac muscle stiffness: a shear wave is generated every 7.5 ms and its
propagation is imaged for 7.5 ms following the generation of the shear wave in the left ventricle.
Supplemental Figure 3: Myocardial stiffness of one control heart as a function of heart
rate. Systolic stiffness is shown with white background and diastolic stiffness with gray
background. For each experimental condition, the acquisitions were performed using three
different LV volumes: deflated balloon, moderately and strongly inflated. The bars show the
mean±s.d. for n=5 measures.
Supplemental Figure 4: Response of normal and stenosed hearts to isoproterenol
Systolic shear modulus and (dP/dt)max for different isoproterenol concentrations (10-9 to 10-7 M)
acquired 2 minutes after application of the drug. * indicates p<0.05, † p<0.002, and ns indicates p
= non significant vs. initial state (n=6).
Supplemental Table 1. Baseline measurements at rest (mean ± s.d). ns = non-significant.
Heart/body Heart
Peak systolic Systolic
Diastolic
N
weight ratio
rate
pressure
stiffness
stiffness
(mg/g)
(bpm)
(mmHg)
(kPa)
(kPa)
Sham operated
6
3.7±0.3
204±13
75.0±18.1
9.2±1.5
1.1±0.4
Aortic stenosis
6
6.1±0.9
158±15
72.5±25.4
10.7±2.0
5.3±0.8
0.002
0.02
ns
ns
<0.0001
p-value