Recent advances in stem cell biology have allowed researchers to efficiently produce large numbers of cardiomyocytes from various pluripotent cell sources. Unfortunately, these cells exhibit properties that are characteristics of immature cardiomyocytes such as poor sarcomere organization, limited calcium handling, and reduced cell size and alignment. Specifically, the actin–myosin motor proteins that form sarcomeres within these cardiomyocytes fail to produce large, highly ordered repeating structures that are distinctive for adult myocytes. Instead, these cells produce heterogeneous sarcomeres that vary in thickness, alignment, and level of organization. Additionally, a large number of cardiomyopathies have been linked to mutations in genes encoding for sarcomeric proteins, resulting in disrupted sarcomere organization. This research focuses on a series of algorithms that provide a quantitative analysis technique to characterize the alignment and organization of sarcomere structures within aggregates and single cardiomyocytes. The scanning gradient Fourier transform (SGFT) method incorporates gradient analysis along with fast Fourier transforms to determine regions of sarcomere organization within individual and a population of cells, yielding a quantitative method of determining sarcomere organization and alignment at the sub-cellular scale. The utility of the SGFT technique is also demonstrated for additional applications, such as breast cancer collagen microstructure and neural rosette patterning.

2
Bashyam
,
M. D.
,
Savithri
,
G. R.
,
Kumar
,
M. S.
,
Narasimhan
,
C.
, and
Nallari
,
P.
, “
Molecular genetics of familial hypertrophic cardiomyopathy (FHC)
,”
J. Hum. Genet.
48
(
2
),
55
64
(
2003
).
3
Bayan
,
C.
,
Levitt
,
J. M.
,
Miller
,
E.
,
Kaplan
,
D.
, and
Georgakoudi
,
I.
, “
Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels
,”
J. Appl. Phys.
105
(
10
),
102042
(
2009
).
4
Benjamin
,
E. J.
,
Blaha
,
M. J.
,
Chiuve
,
S. E.
,
Cushman
,
R.
,
Das
,
S. R.
,
Deo
,
R.
,
de Ferranti
,
S. D.
 et al, “
Heart disease and stroke statistics—2017 update: A report from the American Heart Association
,”
Circulation
135
(
10
),
e146
e603
(
2017
).
6
Bogenrieder
,
T.
and
Herlyn
,
M.
, “
Axis of evil: Molecular mechanisms of cancer metastasis
,”
Oncogene
22
(
42
),
6524
6536
(
2003
).
7
Boudaoud
,
A.
,
Burian
,
A.
,
Borowska-Wykręt
,
D.
,
Uyttewaal
,
M.
,
Wrzalik
,
R.
,
Kwiatkowska
,
D.
, and
Hamant
,
O.
, “
Fibriltool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images
,”
Nat. Protoc.
9
,
457
(
2014
).
8
Bray
,
M.-A.
,
Sheehy
,
S.
, and
Parker
,
K.
, “
Sarcomere alignment is regulated by myocyte shape
,”
Cell Motil. Cytoskel.
65
(
8
),
641
651
(
2008
).
9
Bredfeldt
,
J. S.
,
Liu
,
Y.
,
Pehlke
,
C. A.
,
Conklin
,
M. W.
,
Szulczewski
,
J. M.
,
Inman
,
D. R.
,
Keely
,
P. J.
,
Nowak
,
R. D.
,
Mackie
,
T. R.
, and
Eliceiri
,
K. W.
, “
Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer
,”
J. Biomed. Opt.
19
,
16007
(
2014
).
11
Chang
,
C.
and
Werb
,
Z.
, “
The many faces of metalloproteases: Cell growth, invasion, angiogenesis and metastasis
,”
Trends Cell Biol.
11
,
S37
43
(
2001
).
12
Chen
,
G.
,
Gulbranson
,
D.
,
Hou
,
Z.
,
Bolin
,
J.
,
Ruotti
,
V.
,
Probasco
,
M. D.
,
Smuga-Otto
,
K.
,
Howden
,
S. E.
,
Diol
,
N. R.
,
Propson
,
N. E.
 et al, “
Chemically defined conditions for human iPSC derivation and culture
,”
Nat. Methods
8
,
424
429
(
2011
).
14
Conklin
,
M. W.
,
Eickhoff
,
J. C.
,
Riching
,
K. M.
,
Pehlke
,
C. A.
,
Eliceiri
,
K. W.
,
Provenzano
,
P. P.
,
Friedl
,
A.
, and
Keely
,
P. J.
, “
Aligned collagen is a prognostic signature for survival in human breast carcinoma
,”
Am. J. Pathol.
178
,
1221
1232
(
2011
).
15
Cooley
,
J.
and
Tukey
,
J.
, “
An algorithm for the machine calculation of complex Fourier series
,”
Math. Comput.
19
,
297
301
(
1965
).
17
Dobesh
,
D. P.
,
Konhilas
,
J. P.
, and
de Tombe
,
P. P.
, “
Cooperative activation in cardiac muscle: Impact of sarcomere length
,”
Am. J. Physiol-Heart C
282
,
H1055
H1062
(
2002
).
18
Falzon
,
G.
,
Pearson
,
S.
, and
Murison
,
R.
, “
Analysis of collagen fibre shape changes in breast cancer
,”
Phys. Med. Biol.
53
,
6641
6652
(
2008
).
19
Ferrans
,
V. J.
,
Morrow
,
A. G.
, and
Roberts
,
W. C.
, “
Myocardial ultrastructure in idiopathic hypertrophic subaortic stenosis: A study of operatively excised left ventricular outflow tract muscle in 14 patients
,”
Circulation
45
,
769
792
(
1972
).
20
Francalanci
,
P.
,
Gallo
,
P.
,
Bernucci
,
P.
,
Silver
,
M. D.
, and
D’Amati
,
G.
, “
The pattern of desmin filaments in myocardial disarray
,”
Hum. Pathol.
26
,
262
266
(
1995
).
21
Frisch
,
K. E.
,
Duenwald-Kuehl
,
S. E.
,
Kobayashi
,
H.
,
Chamberlain
,
C. S.
,
Lakes
,
R. S.
, and
Vanderby
, Jr.,
R.
, “
Quantification of collagen organization using fractal dimensions and Fourier transforms
,”
Acta Histochem.
114
,
140
144
(
2012
).
22
Gannier
,
F.
,
Bernengo
,
J. C.
,
Jacquemond
,
V.
, and
Darnier
,
D.
, “
Measurements of sarcomere dynamics simultaneously with auxotonic force in isolated cardiac cells
,”
IEEE Trans. Bio-Med. Eng.
40
,
1226
1232
(
1993
).
23
Hong
,
L.
,
Wan
,
Y.
, and
Jain
,
A.
, “
Fingerprint image enhancement: Algorithm and performance evaluation
,”
IEEE Trans. Pattern Anal.
20
,
777
789
(
1998
).
24
Jacot
,
J.
,
McCulloch
,
A.
, and
Omens
,
J.
, “
Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes
,”
Biophys. J.
95
,
3479
3487
(
2008
).
25
Kabir
,
M. M.
,
Inavalli
,
V. V. G. K.
,
Lau
,
T.-Y.
, and
Toussaint
, Jr.,
K. C.
, “
Application of quantitative second-harmonic generation microscopy to dynamic conditions
,”
Biomed. Opt. Express
4
,
2546
2554
(
2013
).
26
Karlon
,
W. J.
,
Covell
,
J. W.
,
McCulloch
,
A. D.
,
Hunter
,
J. J.
, and
Omens
,
J. H.
, “
Automated measurement of myofiber disarray in transgenic mice with ventricular expression of ras
,”
Anat. Rec.
252
,
612
625
(
1998
).
27
Karlon
,
W. J.
,
McCulloch
,
A. D.
,
Covell
,
J. W.
,
Hunter
,
J. J.
, and
Omens
,
J. H.
, “
Regional dysfunction correlates with myofiber disarray in transgenic mice with ventricular expression of ras
,”
Am. J. Physiol-Heart C.
278
,
H898
H906
(
2000
).
28
Kartasalo
,
K.
,
Pölönen
,
R.-P.
,
Ojala
,
M.
,
Rasku
,
J.
,
Lekkala
,
J.
,
Aalto-Setälä
,
K.
, and
Kallio
,
P.
, “
Cytospectre: A tool for spectral analysis of oriented structures on cellular and subcellular levels
,”
BMC Bioinformatics
16
,
344
(
2015
).
29
Kattman
,
S.
,
Witty
,
A.
,
Gagliardi
,
M.
,
Dubois
,
N.
,
Niapour
,
M.
,
Hotta
,
A.
, and
Keller
,
G. M.
, “
Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines
,”
Cell Stem Cell
8
,
228
240
(
2011
).
31
Kobirumaki-Shimozawa
,
F.
,
Oyama
,
K.
,
Shimozawa
,
T.
,
Mizuno
,
A.
,
Ohki
,
T.
,
Terui
,
T.
,
Minamisawa
,
S.
,
Ishiwata
,
S.
, and
Fukuda
,
N.
, “
Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function
,”
J. Gen. Physiol.
147
,
53
62
(
2016
).
33
Laflamme
,
M. A.
,
Chen
,
K. Y.
,
Naumova
,
A. V.
,
Muskheli
,
V.
,
Fugate
,
J. A.
,
Dupras
,
S. K.
,
Reinecke
,
H.
,
Xu
,
C.
,
Hassanipour
,
M.
,
Police
,
S.
 et al, “
Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts
,”
Nat. Biotechnol.
25
,
1015
1024
(
2007
).
34
Laflamme
,
M. A.
and
Murry
,
C. E.
, “
Regenerating the heart
,”
Nat. Biotechnol.
23
,
845
856
(
2005
).
35
Lian
,
X.
,
Hsiao
,
C.
,
Wilson
,
G.
,
Zhu
,
K.
,
Hazeltine
,
L. B.
,
Azarin
,
S. M.
,
Raval
,
K. K.
,
Zhang
,
J.
,
Kamp
,
T. J.
, and
Palecek
,
S. P.
, “
Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling
,”
Proc. Natl. Acad. Sci. U.S.A.
109
(
27
),
E1848
E1857
(
2012
).
36
Lippmann
,
E. S.
,
Estevez-Silva
,
M. C.
, and
Ashton
,
R. S.
, “
Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors
,”
Stem Cells
32
,
1032
1042
(
2014
).
37
Lundy
,
S.
,
Zhu
,
W.
,
Regnier
,
M.
, and
Laflamme
,
M. A.
, “
Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells
,”
Stem Cells Dev.
22
,
1991
2002
(
2013
).
38
Mandenius
,
C.-F.
,
Steel
,
D.
,
Noor
,
F.
,
Meyer
,
T.
,
Heinzle
,
E.
,
Asp
,
J.
,
Arain
,
S.
,
Kraushaar
,
U.
,
Bremer
,
S.
,
Class
,
R.
 et al, “
Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: A review
,”
J. Appl. Toxicol.
31
,
191
205
(
2011
).
39
Mansour
,
H.
,
de Tombe
,
P. P.
,
Samarel
,
A. M.
, and
Russell
,
B.
, “
Restoration of resting sarcomere length after uniaxial static strain is regulated by protein kinase Cε and focal adhesion kinase
,”
Circ. Res.
94
,
642
649
(
2004
).
40
Mardia
,
K. V.
and
Jupp
,
P. E.
,
Directional Statistics
(
John Wiley & Sons
,
New York
,
2000
).
41
Maron
,
B. J.
,
McKenna
,
W. J.
,
Danielson
,
G. K.
,
Kappenberger
,
L. J.
,
Kuhn
,
H. J.
,
Seidman
,
C. E.
,
Shah
,
P. M.
,
Spencer
,
W. H.
,
Spirito
,
P.
,
Ten Cate
,
F. J.
 et al, “
American College of Cardiology/European Society of Cardiology clinical expert consensus document on hypertrophic cardiomyopathy
,”
J. Am. Coll. Cardiol.
42
,
1687
1713
(
2003
).
43
Nectow
,
A.
,
Kilmer
,
M. E.
, and
Kaplan
,
D. L.
, “
Quantifying cellular alignment on anisotropic biomaterial platforms
,”
J. Biomed. Mater. Res. A
102
,
420
428
(
2014
).
46
Provenzano
,
P. P.
,
Eliceiri
,
K. W.
,
Campbell
,
J. M.
,
Inman
,
D. R.
,
White
,
J. G.
, and
Keely
,
P. J.
, “
Collagen reorganization at the tumor-stromal interface facilitates local invasion
,”
BMC Med.
4
,
38
(
2006
).
47
Rao
,
R. A. R.
,
Mehta
,
M. R.
, and
Toussaint
, Jr.,
K. C.
, “
Fourier transform-second-harmonic generation imaging of biological tissues
,”
Opt. Express
17
,
14534
14542
(
2009
).
49
Robertson
,
C.
,
Tran
,
D.
, and
George
,
S.
, “
Concise review: Maturation phases of human pluripotent stem cell-derived cardiomyocytes
,”
Stem Cells
31
,
829
837
(
2013
).
50
Salick
,
M. R.
,
Napiwocki
,
B. N.
,
Sha
,
J.
,
Knight
,
G. T.
,
Chindhy
,
S. A.
,
Kamp
,
T. J.
,
Ashton
,
R. S.
, and
Crone
,
W. C.
, “
Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes
,”
Biomaterials
35
,
4454
4464
(
2014
).
51
Sottile
,
J.
, “
Regulation of angiogenesis by extracellular matrix
,”
Biochim. Biophys. Acta Rev. Cancer
1654
,
13
22
(
2004
).
54
Toepfer
,
C. N.
,
Sharm
,
A.
,
Cicconet
,
M.
,
Garfinkel
,
A. C.
,
Mücke
,
M.
,
Neyazi
,
M.
,
Willcox
,
J. A. L.
,
Agarwal
,
R.
,
Schmid
,
M.
,
Rao
,
J.
 et al, “
Sarctrack: An adaptable software tool for efficient large-scale analysis of sarcomere function in hiPSC-cardiomyocytes
,”
Circ. Res.
124
,
1172
1183
(
2019
).
55
Willems
,
E.
,
Spiering
,
S.
,
Davidovics
,
H.
,
Lanier
,
M.
,
Xia
,
Z.
,
Dawson
,
M.
,
Cashman
,
J.
, and
Mercola
,
M.
, “
Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell–derived mesoderm
,”
Circ. Res.
109
,
360
364
(
2011
).
57
Yang
,
L.
,
Soonpaa
,
M.
,
Adler
,
E.
,
Roepke
,
T. K.
,
Kattman
,
S. J.
,
Kennedy
,
M.
,
Henckaerts
,
E.
,
Bonham
,
K.
,
Abbott
,
G. W.
,
Linden
,
R. M.
 et al, “
Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population
,”
Nature
453
,
524
528
(
2008
).
58
Yang
,
X.
,
Pabon
,
L.
, and
Murry
,
C. E.
, “
Engineering adolescence: Maturation of human pluripotent stem cell-derived cardiomyocytes
,”
Circ. Res.
114
(
3
),
511
523
(
2014
).
60
Zhang
,
J.
,
Klos
,
M.
,
Wilson
,
G. F.
,
Herman
,
A. M.
,
Lian
,
X.
,
Raval
,
K. K.
,
Barron
,
M. R.
,
Hou
,
L.
,
Soerens
,
A. G.
,
Yu
,
J.
 et al, “
Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: The matrix sandwich method
,”
Circ. Res.
111
,
1125
1136
(
2012
).

Supplementary Material

You do not currently have access to this content.