Conceived and designed the experiments: LM AB AGP. Performed the experiments: LM AB. Analysed and interpreted the data: LM. Drafted and edited the manuscript: LM. Contributed analysis tools: AB. Participated in the writing and editing of the manuscript: AB AGP JWB.
Current address: School of Applied Sciences, Bournemouth University, Talbot Campus, Poole, United Kingdom
The authors have declared that no competing interests exist.
Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows
With a simple heart, a transparent body surface at larval stages and available genetic tools we chose
These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in
Dilated cardiomyopathy (DCM) is a progressive pathological cardiac condition characterized by an enlarged heart with impaired contractility, defects that often lead to heart failure. To date a wide range of genes, from those involved in mechanobiochemical signaling to components of the contractile architecture, have been confirmed to cause DCM when defective
The fruitfly,
Key to the potential of this
Here we describe an OCT instrument which can operate in two regimes. In the imaging regime, the system can acquire OCT images, with either cross sectional or
Initially we applied
The larval heart chamber possesses three pairs of laterally located ostia along its axis
Adult
In addition, although the same proportion of
Co`ntrol ( |
||
n |
n = 47 | |
Wavy contraction | 64 | 62 |
Occasional chamber shortening | 28 | − |
Dominant chamber shortening | − | 27 |
Chamber shortening only | 0 | 4 |
Pauses | 8 | 34 |
n = 27 | n = 27 | |
Regular rate | 82 | 63 |
Irregular rate | 11 | 11 |
Pause (>2 sec) | 7 | 26 |
*: number of animal used.
It is well documented that the fruitfly heartbeat is irregular. It has been noticed previously that short periods of heart pausing are associated with eating activity and with preparation for crawling in
The following information was generated from the Doppler audio recording. Top panel is heart rate in 1 minute. In 10 sec heart rate counting group, Mean ± SEM for controls is 154±12.09 (n = 25), for
Heart arrhythmia is associated with DCM-causing tropomyosin mutations in humans, but has not been apparent in the adult
As shown in the bottom panels of
Amplitude recorded by the Audicity plot (
In order to improve the sensitivity of the methodologies used to dissect the genetic networks underlying dilated cardiomyopathy, we built a dual-regime OCT instrument dedicated for the
The reduced light reflectivity in the
Our data reaffirms that, predominantly, heart contraction takes the form of a peristaltic wave of posterior origin. In embryos
Arrhythmias occur in one third of the patients suffering from DCM associated with mutation in the α-tropomyosin gene
The ideal instrument for recording cardiac dynamics in
OCT has several of these attributes. It is a label-less imaging technique that generates an image from interference between an internal reference beam and light reflected by the target. It is capable of imaging at depth through the cuticle.
Previous studies that applied OCT to imaging of the adult
Although the 5 Hz acquisition rate has largely improved our capability of following the heart rate, we are still missing important parts of the heart wall motion during each beat. To address this deficiency, we complemented the information acquired during the imaging process with that acquired during Doppler signal recording. In this regime, the heart wall motion is recorded in real time. This technique exploits the fact that a moving light-scattering object imposes a Doppler shift to the frequency of the scattered light that is proportional to the velocity of the movement of the object. In our instrument a Doppler shift was produced when the distance between fixed detecting focal point and heart wall changes due to the contraction/relaxation movement of the latter. The wavelength of the SLD light source, together with the speed of movement of the heart wall, place the Doppler frequency within the audio range; hence our instrument performs like a stethoscope and the Doppler signal may be plotted as a sound signal using Audicity software. A typical heartbeat audio recording from a control larva is shown in the supplement material (
A schematic diagram of the dual
Light back-scattered by the specimen passes a second time through the object arm, guided via the first directional coupler towards the second single mode directional coupler where it interferes with that coming from the reference arm. Both output fibers from the second coupler are connected to two pin photo-detectors, PD1 and PD2, in a balanced photo-detection unit constructed using a differential amplifier, DA. The OCT signal is rectified and low pass filtered in the demodulator DMOD. A computer-driven translation stage, TS1, is used to alter the reference path length to select different depths for C-scans while acquiring stacks of C-scans as well as scanning the depth in the B-scan acquisition mode. The scanning procedure is similar to that used in any confocal microscope, where the fast scanning is
To switch the system to the audio (stethoscope) regime, the scanning of the beam across the specimen is interrupted using switches SWX and SWY. Hence, the recorded interferometric signal, low-pass filtered by the DMOD block, is exclusively due to projection of movement of the heart wall along the axial direction of the objective lens.
The
To image heart activity,
The axial plane at which the heart exhibited its widest diameter was chosen for imaging. The photodetected optical signal was finally converted into images by means of a custom Bitflow SDK based software operating on a Raven BitFlow dual stage frame grabber. Individual stacks of over 100 frames per larva were recorded. After imaging, the system was switched to the stethoscope Doppler OCT regime for audio recording. Immediately after switching, the specimen carrying stage was slightly readjusted till the position where the highest pitch or frequency of the Doppler signals was detected. Then the Doppler signal in that position in each heart was acquired via the audio card of the computer and plotted using Audacity software (
The heart chamber size, the distance between the two sides of heart walls in the heart image frames at the sagittal plane was measured with Image J (
The heartbeat tracing in
Student's t test was used in the statistic analysis.
Sound of
(3.08 MB WAV)
Heart beat in a wild type
(5.23 MB AVI)
The speed of the movie is 5 frames/ sec. Cardiac chamber-shortening contraction in a Tropomyosin mutant larva.
(6.51 MB AVI)
Cardiac pausing in a Tropomyosin mutant larva. The speed of the movie is 5 frames / sec.
(5.09 MB AVI)