Key topics covered during this webinar include: Evaluating cardiac contractility using mean or peak aortic acceleration Investigating cardiac relaxation using mitral peak early velocity to peak atrial velocity ratio Interpreting myocardial perfusion capacity through coronary flow reserve at baseline and with disease or other conditions How Doppler Flow Velocity measurements can be used in translational research from mice to mammals In a recent ground-breaking publication in Scientific Reports by Nature Research, Perez et al. highlight the use of noninvasive blood flow velocity measurements to quantify cardiac contractility as a surrogate to +dP/dt max. The article titled “Aortic acceleration as a noninvasive index of left ventricular contractility in the mouse” describes an alternate methodology to what is highly considered the gold standard for evaluating cardiac contractility and relaxation in preclinical research. The acute and terminal nature of acquiring +dP/dt using invasive blood pressure catheters is less than ideal, so finding a noninvasive surrogate is of great interest to the scientific research community. Utilizing a Doppler Flow Velocity System (DFVS) from Indus Instruments, Dr. Reddy and his group show that peak acceleration in the ascending aorta can be used in place of invasive LVP catheters. This novel technique enables serial measurements in the same animal, which reduces animal-to-animal variability, allows for the use of fewer subjects, and decreases data collection time. Please join us during our upcoming webinar on March 4th, 2021 at 11am EST to hear Dr. Reddy present his findings with a LIVE Q&A session at the end. References: Perez, J.E.T., Ortiz-Urbina, J., Heredia, C.P. et al. Aortic acceleration as a noninvasive index of left ventricular contractility in the mouse. Sci Rep 11, 536 (2021)

1. Aortic Acceleration: A Noninvasive
Alternative to +dP/dtmax
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2. Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
Aortic Acceleration: A Noninvasive
Alternative to +dP/dtmax

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4. Audience Poll

5. Assessment of Cardiac Function Using
Noninvasive Blood Flow Velocity Measurements
Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
Aortic Acceleration: A Noninvasive
Alternative to +dP/dtmax

6. Methodology
• Pulsed Doppler ultrasound
- Why is it needed?
- How does it work?
Applications
• Cardiac systolic measurement
- Acceleration vs. +dP/dtmax
• Other cardiovascular measurements
Presentation Outline

7. Most CV measurements and
parameters are functions of
time, so we need waveforms
• Rodents are animals of choice in basic research
• Genetic, surgical, pharmacological manipulations
• Alterations in cardiovascular system
• Need cardiovascular phenotyping
Small Animal Noninvasive Cardiovascular Phenotyping

8. Most CV measurements and
parameters are functions of
time, so we need waveforms
“Have a nice day
at the lab, dear?”
But, the challenge is
to be Noninvasive
• Rodents are animals of choice in basic research
• Genetic, surgical, pharmacological manipulations
• Alterations in cardiovascular system
• Need cardiovascular phenotyping
Small Animal Noninvasive Cardiovascular Phenotyping

9. 9
Advantages
• Noninvasive - longitudinal studies
• Short signal acquisition times
• Can be measured at various locations
• Possible to achieve small angles
Know-how
• Knowledge of anatomy
• Shapes and timing of waveforms
Pulsed Doppler Ultrasound
“Not an echocardiography system”

10. Methodology – Doppler Flow Velocity

11. Pulsed Doppler Ultrasound: How does it work?
Relationship
between blood
velocity & Doppler
shift is given as:
V = (c Δf)/(2fo cos θ)
where…
V = flow velocity (cm/sec)
c = velocity of sound (cm/sec)
Δf = Doppler shift (Hz)
fo = transmission frequency (Hz)
θ = angle between velocity
vector & beam vector
θ artery

12. Why Blood Flow Velocity ?

13. Scaling in Mammals from Elephants to Mice
General allometric equation: Y = a.BWb
Parameter Relationship to BW (kg)* Value (BW=0.025kg)
Heart weight (mg) a BW1 4.3 BW 112 mg
LV volume (μl) a BW1 2.25 BW 56 ml
Stroke volume (μl) a BW1 0.95 BW 24 ml
Heart rate (bpm) a BW-1/4 230 BW-1/4 578 bpm
Cardiac output (ml/min) a BW3/4 224 BW3/4 14 ml/min
Aortic diameter (mm) a BW3/8 3.6 BW3/8 0.9 mm
Arterial pressure (mmHg) a BW0 100 100 mmHg
Aortic velocity (cm/s) a BW0 100 100 cm/s
PW velocity (cm/s) a BW0 500 500 cm/s
*T.H. Dawson, “Engineering design of the cardiovascular system of mammals,” Prentice Hall, 1991.

14. Scaling in Mammals from Elephants to Mice
General allometric equation: Y = a.BWb
Parameter Relationship to BW (kg)* Value (BW=0.025kg)
Heart weight (mg) a BW1 4.3 BW 112 mg
LV volume (μl) a BW1 2.25 BW 56 ml
Stroke volume (μl) a BW1 0.95 BW 24 ml
Heart rate (bpm) a BW-1/4 230 BW-1/4 578 bpm
Cardiac output (ml/min) a BW3/4 224 BW3/4 14 ml/min
Aortic diameter (mm) a BW3/8 3.6 BW3/8 0.9 mm
Arterial pressure (mmHg) a BW0 100 100 mmHg
Aortic velocity (cm/s) a BW0 100 100 cm/s
PW velocity (cm/s) a BW0 500 500 cm/s
*T.H. Dawson, “Engineering design of the cardiovascular system of mammals,” Prentice Hall, 1991.

15. Doppler Flow Velocity and
Rodent Monitoring Systems

16. Set-up for Noninvasive Doppler
Measurements in Mice

17. • Maintain anesthesia
• Monitor ECG and respiration
• Monitor body temperature
• Maintain board or body temperature
• Perform noninvasive measurements
• Perform surgery
• Perform invasive measurements
ECG
Respiration
With this configuration we can:
RA LA
LL
RL
ECG/Resp
Electrodes
Mouse ECG &
Warming Pad
Warming
Zone
ECG/Resp Amplifier Temp Control

18. Application – Cardiac Systolic Function

19. Mouse Cardiac Doppler Signals - Aortic velocity
Using 10 MHz
Doppler Probe
ECG
Aortic Velocity

20. Cardiac Systolic Parameters
ao ac
R-R Interval = 156 ms
Aop
Systole Diastole
t1
Aop – Peak aortic flow velocity
t1 – Pre-ejection time
t2 – Aortic ejection Tine
t3 – Time to peak velocity (Rise time)
Aortic Outflow Waveform
ao – Aortic valve opens
ac – Aortic valve closes
t2
ECG
t3
Measurements/parameters:
• Heart Rate (from R-R)
• Pre-ejection time
• Rise time
• Ejection time
• Peak Velocity
• Mean Velocity
• Peak Acceleration
• Mean Acceleration
• Stroke Distance

21. Cardiac Systolic Function: Peak Aortic Velocity
Taffet et al., J Geron
Biol Sci 52A, 1997.
Reddy et al., J Geron
Biol Sci 62A, 2007.
Taffet et al., Am J
Physiol 270, 1996.
Mayr et al., Physiol
Rep 4, e12765, 2016.
Reddy et al., IEEE
TBME 52, 2005.
DeLaughter et al.,
FASEB J 13, 1999.
Hartley et al., Am J
Physiol 279, 2000.

22. Cardiac Systolic Function: Aortic Velocity Examples
Robinson et al., BioMed Res Intl, #645153, 2015
Sham Aprepitant
Preteated
Kelsey et al., PLoS Genetics, 9, 2013
Wild type Klf3H275R/+ mice
cm/s
Cieslik et al., J Molec Cell Cardiol, 63, 2013
Saline AICAR
This is an example of murine myocarditis
caused by encephalomyocarditis virus
(EMCV) and pretreatment with Aprepitant
improves heart function as observed by
increased peak aortic flow velocity
Example of the ascending aortic blood velocity
waveforms in a WT mouse with normal peak aortic
velocity and in a mouse with a missense mutation in the
klf3 gene which has a high peak velocity trait that they
used to identify mutants .
Example showing AICAR-dependent
AMPK activation prevents adverse
remodeling after MI. Systolic dysfunction
was prevented by AICAR treatment. This
represent changes in the heart function
at 4wks post-MI compared to pre-values.

23. Audience Poll

24. Cardiac Contractility – Acceleration vs. +dP/dtmax

25. Mouse Cardiac Contractility and Relaxation
Aortic Velocity
LV Pressure (P) First Derivative of LV Pressure - dP/dt
dP
+―
dtmax
dP
– ―
dtmax
“Invasive & Terminal”

26. Invasive LV Pressure Measurement in the Mouse
• It’s a terminal study; acute study only
• Aortic regurgitation
• Perturbation of hemodynamics
- cannulation of carotid artery
- straightening of the asc. aorta
- straightening of the heart
• Atherosclerosis or other mouse model
with stiffer arteries may be problematic

27. Ranking of Indices of LV Contractility by Lambert et al.
• The top 7 most sensitive indices require
invasively measured LV pressure.
• The next 2 most sensitive indices (mean &
peak aortic acceleration) require non-
invasively measured aortic blood velocity.
• Followed by dP/dtmax which again requires
the invasively measured LV pressure.
Lambert CR Jr, Nichols WW, Pepine CJ. Indices of
ventricular contractile state: comparative sensitivity and
specificity. Am Heart J. 106, 136-144, 1983.

28. Hunt et al., Cathet Cardiovasc Diagn 23, 1991
Peak dP/dt = 74.2V2/T + 847
R = 0.77
2500
2000
1500
1000
0 5 10 15 20 25
Peak
dP/dt
(mmHg
s
-1
)
V2/T (m2 s-3)
Not evaluated for various loading conditions
Noninvasive Assessment of LV contractility - Dogs & Patients
Harada et al., Heart Vessels 3, 1987
Dogs
800
600
400
200
0
0 200 400 600 800
ρc
Max
du/dt
(kPa/s)
Max dP/dt (kPa/s)
Y = 1.01X - 2
R = 0.97
Patients

29. Noninvasive Assessment of LV Contractility - Sheep
Bauer et al., JACC 40, 2002
Aortic acceleration (LVOTAcc) vs. LV maximal elastance ( Em)
 - various loading conditions;  - acute coronary occlusion
Good correlation between LVOTAcc and LV +dP/dt (r = 0.62)
LV maximal elastance ( Em)
Aortic acceleration (LVOTAcc)

30. In the Mouse: Aortic Flow Velocity & LV Pressure
Tovar Perez, et al. Scientific Reports, 11:536, 2021.

31. Aortic outflow velocity (V) & its derivative (dV/dt) and
Left ventricular pressure (P) & its derivative (dP/dt)
Tovar Perez, et al. Scientific Reports, 11:536, 2021.

32. Noninvasive surrogate measurements for P’ (peak +dP/dt)
derived from Doppler aortic blood flow velocity waveform
Tovar Perez, et al. Scientific Reports, 11:536, 2021.

33. Noninvasive Measurements: DFVS and Echocardiography
Aortic Blood Flow Velocity & Related Parameters
Tovar Perez, et al. Scientific Reports, 11:536, 2021.
Parameter
Doppler
No angle
correction
Echocardiography
Angle correction
None 45° correction
t-test
Dopp. vs.
Echo (0°)
t-test
Dopp. vs.
Echo (45°)
vp (cm/s) 90.2±4.4 58.2±8.1 82.3±11.5 p<0.05 p=NS
vp
2/T (m2/s3) 59.3±10.1 27.6±8.5 55.2±16.9 p<0.05 p=NS
v’p (cm/s2) 6449±1609 3672±880 5191±1245 p<0.05 p=NS
v’m (cm/s2) 12986±1609 7371±1609 10421±2591 p<0.05 p=NS
Simultaneous noninvasive measurements:
echcocardiography & pulsed Doppler flow velocity

34. Mouse Studies: Acceleration Examples
(Vincelette et al., Translational Research, vol.148, 2006)

35. Weisleder et al., PNAS, 101, 2004
Bcl-2 overexpression prevents decline in
cardiac function in desmin null mice
Cieslik et al., J Molec Cell Cardiol, 63, 2013
Cardiac Systolic Function: Acceleration Examples
Saline AICAR
Example 1
Example 2

36. 100
80
60
40
20
0
peak
aortic
velocity
(cm/s)
7000
6000
5000
4000
3000
2000
1000
0
mean
acceleration
(cm/s
2
)
Rieneke et al., PLoS ONE, 7:e53395, 2012
Cardiac Systolic Function: Acceleration Examples
Example 3

37. Other Cardiovascular Measurements

38. Mouse Cardiac Doppler Signals – Mitral Velocity
Using 10 MHz
Doppler Probe
ECG
Mitral Velocity

39. Cardiac Diastolic Function
mc
ao ac
mo mc
R-R Interval = 161 ms
Ep
Ap
Systole Diastole
t1 t2 t3
t5
t6
t4
t8
Ep – Peak early flow velocity
Ap – Peak atrial flow velocity
t1 – Isovolumic contraction time
t2 – Isovolumic relaxation time
t3 – Duration of early flow velocity (EFV)
t4 – Acceleration time of EFV
t5 – Deceleration time of EFV
t6 – Time from Ep-½Ep
t7 – Linear deceleration time of EFV
t8 – Duration of atrial flow velocity
mc – mitral valve closes
ao – Aortic valve opens
ac – Aortic valve closes
mo – Mitral valve opens
t7
ECG
Mitral Inflow Waveform

40. Cardiac Diastolic Function
mc
ao ac
mo mc
R-R Interval = 161 ms
Ep
Ap
Systole Diastole
t1 t2 t3
t5
t6
t4
t8
Mitral Inflow Waveform
t7
ECG
Measurements/parameters:
• E-Time Duration
• E-Acceleration Time
• E-Deceleration Time
• E-Peak to ½ E-Peak Time
• E-Linear Deceleration Time
• A-Time Duration
• Isovolumic Contraction Time
• Isovolumic Relaxation Time
• E-Peak Velocity
• E-Stroke Distance
• E-Linear Deceleration Rate
• A-Peak Velocity
• A-Stroke Distance
• E-A Peak Velocity Ratio

41. Cardiac Diastolic Function
÷ =

42. Doppler Signals from Several Arterial Sites
Hartley et al., ILAR J 43:147-8, 2002

43. Noninvasive coronary Doppler signals from a mouse anesthetized at low
and high levels of isoflurane gas to measure coronary flow reserve
-90-
-
-
-60-
-
-
-30-
-
cm/s
- 0 -
| 400 ms |
24-
16-
8-
kHz
0-
ECG HR = 450
Vlow
low =1.0% high =2.5%
CFR = H/B = Vhigh/Vlow = 4.2
HR = 465
Hartley et al., Ultrasound Med Biol, 33, 2007
Vhigh

44. Aortic
band
-500
cm/s
-0
-20
-0
-160
cm/s
-0
ECG
Aortic Arch Jet Velocity - 10 MHz Doppler
Left Carotid Artery Velocity - 20 MHz Doppler
Right Carotid Artery Velocity - 20 MHz Doppler
msec
ΔP~75 mmHg
mm scale
Flow
velocity
measurements
to
quantify
response
to
banding
(TAC)
in
mice
Hartley et al., Ultrasound Med Biol 34, 2008

45. Coronary
flow
reserve
in
a
banded
mouse
Hartley et al., Ultrasound Med Biol 34, 2008

46. Pulse-Wave Velocity Measurements in Mice
PWV measured from signals acquired non-simultaneously
from aortic arch and abdominal aortic sites
PWV measured from signals acquired simultaneously from
aortic arch and abdominal aortic sites

47. Summary
 Cardiac systolic function (aortic FV- LV contractility)
 Cardiac diastolic function (mitral FV - LV relaxation)
 Myocardial perfusion index (coronary FV - CFR)
 Pressure overload by TAC (cardiac and coronary reserve)
 Pulse Wave Velocity (aortic/arterial stiffness)
 Noninvasive - allows for serial studies
 Measurements at very small angles
 Short signal acquisition times
 Can be measured at various locations
 Replaces invasive measurements
 Not echocardiography

48. Acknowledgements
Craig Hartley
Lloyd Michael
George Taffet
Mark Entman
Yong Xu
Thuy Pham
Celia Pena Heredia
Jorge Enrique Tovar Perez
Jesus Ortiz-Urbina
Jennifer Pocius
Jim Brooks
Ross Hartley
Technicians: Faculty Collaborators:
Sridhar Madala - Indus Instruments
Yi-Heng Li - NCK University, Taiwan
Jim Wang - Berlex Biosciences (now at Crown Biosciences)
Rochelle Buffenstein - UT San Antonio (now at Calico Labs)

49. Q&A
Session:
Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
areddy@bcm.edu
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