UEF//eRepository
DSpace https://erepo.uef.fi
Rinnakkaistallenteet Terveystieteiden tiedekunta
2017
The follow-up of progressive
hypertrophic cardiomyopathy using magnetic resonance rotating frame relaxation times
Khan MA
Wiley-Blackwell
info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
© John Wiley & Sons Ltd All rights reserved
http://dx.doi.org/10.1002/nbm.3871
https://erepo.uef.fi/handle/123456789/5274
Downloaded from University of Eastern Finland's eRepository
1
The follow-up of progressive hypertrophic
2
cardiomyopathy using magnetic resonance
3
rotating frame relaxation times
4
Muhammad Arsalan Khan (muhammad.khan@uef.fi),1 Hanne Laakso 5
(laaks020@umn.edu),2 Svetlana Laidinen (svetlana.laidinen@uef.fi),1 Sanna 6
Kettunen (sanna.kettunen@uef.fi),1 Tommi Heikura (tommi.heikura@uef.fi),1 Seppo 7
Ylä-Herttuala (seppo.ylaherttuala@uef.fi),1,3 and Timo Liimatainen 8
(timo.liimatainen@uef.fi)1,4 9
10 11
[1] Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for 12
Molecular Sciences, University of Eastern Finland, Kuopio, Finland 13
[2] Center for Magnetic Resonance Research, University of Minnesota, Minneapolis, 14
Minnesota, United States of America 15
[3] Heart Center, Kuopio University Hospital, Kuopio, Finland 16
[4] Diagnostic Imaging Center, Kuopio University Hospital, Kuopio, Finland 17
18
Word Count 19
5339 20
21
Keywords 22
Cardiovascular magnetic resonance – Relaxation along fictitious field – HCM 23
Abbreviations:
24
Beff Effective radio frequency field CVF Collagen volume fraction CW Continuous wave
ECG Electrocardiography
HCM Hypertrophic cardiomyopathy HS Hyperbolic secant
LGE Late gadolinium enhancement LV Left ventricle
M Magnetization
PBS Phosphate buffered saline
RRTD Relative relaxation time difference ROI Region of interest
SAR Specific absorption rate
T1ρ Longitudinal rotating frame relaxation time TAC Transverse aortic constriction
TRAFF Relaxation along a fictitious field
25
Abstract 26
Magnetic resonance rotating frame relaxation times appeared as an alternative non- 27
contrast agent choice for chronic myocardial infarct diagnosis. Fibrosis typically 28
occurs in progressive hypertrophic cardiomyopathy. Fibrosis has been imaged in 29
myocardial infarcted tissue using rotating frame relaxation times, which provides the 30
possibility to follow up progressive cardiomyopathy without contrast agents.
31
Mild and severe left ventricular hypertrophy was induced to mice by transverse aortic 32
constriction, and the longitudinal rotating frame relaxation times (T1ρ) and relaxation 33
along the fictitious field (TRAFF2, TRAFF3)) were measured at 5, 10, 24 62 and 89 days 34
after transverse aortic constriction in vivo. Myocardial fibrosis was verified using 35
Masson’s trichrome staining.
36
Increases in the relative relaxation time differences of T1ρ along with TRAFF2 and 37
TRAFF3 between fibrotic and remote tissues over time were observed. Furthermore, 38
TRAFF2 and TRAFF3 showed higher relaxation times overall in fibrotic tissue than T1ρ. 39
Relaxation time differences were highly correlated with an excess of histologically 40
verified fibrosis.
41
We found that TRAFF2 and TRAFF3 are more sensitive to hypertrophic cardiomyopathy 42
related tissue changes than T1ρ and served non-invasive diagnostic magnetic 43
resonance imaging markers to follow up the progressive hypertrophic cardiomyopathy 44
in mouse model.
45
1. Background 46
The growth of the population, aging and the Western lifestyle has increased the 47
incidence of cardiovascular diseases globally [1]. Hypertrophic cardiomyopathy 48
(HCM) is one of the most common inheritable myocardial diseases [2]; however, it 49
can also be a result of long-lasting high systolic blood pressure. HCM is linked to 50
heart failure and subsequent sudden death, which may be the first symptom of the 51
disease [3]. HCM causes thickening of the left ventricle (LV) and fibrosis in the later 52
stage, and in a small number of patients, it can lead to a dilated LV [3]. The clinical 53
diagnosis of HCM in patients showed an increase in LV wall thickness from mild (13- 54
15 mm) [4-6] to severe (≥30 mm); normal thickness of the myocardium is less than 12 55
mm [7-10].
56
Magnetic resonance imaging (MRI) is an important diagnostic tool for identifying 57
segmental left ventricular hypertrophy that is undetectable by echocardiography [2].
58
Cine MRI enables the evaluation of LV volume, LV mass and LV thickness in HCM 59
[11-14] and is therefore suitable for detecting contractile damage in HCM [15]. Late 60
gadolinium enhancement (LGE)-based imaging is performed to quantify myocardial 61
fibrosis, and the LGE image is typically obtained using a T1-weighted sequence.
62
Fibrotic tissue appears brighter compared to normal myocardium in LGE images [16].
63
In a previous study, in an HCM patient T2-weighted images showed patchy 64
mesocardial edema, which is often smaller in area than fibrotic scar tissue [17].
65
Edema that is visualized by T2-weighted imaging corresponds to inflammation and 66
ischemia [17].
67
A previous study suggested that T1 measurement in patients is a feasible imaging 68
option for identifying damaged myocardium without LGE and provides an alternative 69
imaging method to distinguish normal and abnormal myocardium [18]. T1 mapping 70
can discriminate between HCM and hypertensive myocardial disease. Previously, it 71
was demonstrated that native T1 mapping could detect early changes in the 72
myocardium of patients with HCM [19]. The presence of fibrosis in HCM resulted in 73
an abnormal myocardium [20, 21]. Longer T1 values were found in abnormal 74
myocardium with scar tissue than in the remote myocardium in HCM; however, after 75
the administration of contrast agent, the myocardium with diffuse scar tissue showed 76
a shorter T1 relaxation time and delayed normalization of T1 with washout of 77
gadolinium [22]. These studies demonstrated the potential of T1 mapping for the 78
characterization of fibrosis in HCM.
79
Longitudinal relaxation during radio frequency (RF) pulses is characterized by the 80
rotating frame relaxation with the relaxation time constant T1 in the rotating frame 81
(T1ρ). Rotating frame relaxations are sensitive to slow molecular motions from dipolar 82
interactions and other molecular processes occurring close to the frequencies of the 83
RF irradiation. Typically, these frequencies are from a few hundred to a few thousand 84
Hz, depending on the experimental setup. T1ρ describes the relaxation along Beff, 85
where Beff is a geometrical sum of the RF field B1 and off-resonance field components.
86
Continuous wave (CW) T1ρ is typically measured by applying a composite RF pulse 87
consisting of a 90° hard pulse or an adiabatic half-passage pulse to flip magnetization 88
(M) to the xy-plane, a CW pulse to spin-lock M, and a 90° flip to return M along the 89
z-axis (||B0), as described previously [23-25]. CW T1ρ appeared to attain a similar 90
spatial distribution to LGE in chronic myocardial infarct in a porcine model [23].
91
Because T1ρ depends on the RF amplitude, molecular processes occurring at low 92
frequencies may suppress the contrast between fibrotic and remote myocardium when 93
CW with a low amplitude (below 500 Hz) is used. It is suggested that problems can 94
be avoided by using moderate CW pulse amplitudes (over 500 Hz) [23].
95
Higher specific absorption rates (SAR) related to the CW T1ρ measurements limit the 96
clinical applicability of T1ρ with moderate RF powers. A previous study of the human 97
brain showed that SAR can be markedly reduced compared to CW T1ρ by using 98
relaxation along the fictitious field (RAFF) for measuring the rotating frame 99
relaxation [26]. In RAFF, relaxation is measured during sine amplitude- and cosine 100
frequency-modulated RF pulses, and signal decay during the RF pulse is 101
characterized by the relaxation time constant TRAFF. In contrast to the T1ρ
102
measurement, the RAFF-pulse creates an effective field, which consists of Beff and a 103
fictitious field component, along which M is locked [26, 27]. A greater reduction in 104
the SAR can be achieved by applying a RAFF in the nth rotating frame (RAFFn, 105
corresponding relaxation time constant TRAFFn) as shown previously [28]. Relaxation 106
times of TRAFFn are measured byapplying nested sine- and cosine-modulated RF 107
pulses as described elsewhere [28].
108
In this study, we followed up progressive HCM using rotating frame relaxation times 109
(T1ρ, TRAFF2 and TRAFF3). We showed that increased rotating frame relaxation times 110
were associated with the severity of HCM derived from histology sections stained 111
with Masson’s trichrome.
112
2. Methods 113
A total of 16 male (C57BL/6J) mice, 8-12 weeks of age and weighing 25-30 g, were 114
divided into three groups (I-III) as follows: the first group (I) included 6 mice with 115
mild LV hypertrophy; the second group (II) included 5 mice with severe LV 116
hypertrophy; and the third group (III) included 5 control mice with intact hearts. In 117
vivo measurements were performed in all the animals. Measurements for group (I) 118
were performed at 5 time points (5, 10, 24, 62, and 89 days), and for group (II) at 3 119
time points (5, 10, and 24 days) after transverse aortic constriction (TAC). All 120
experiments were performed according to the national guidelines for laboratory 121
animal use and under license ESAVI-2014-005089 of the Finnish National Animal 122
Experiment Board.
123
2.1. TAC surgery 124
TAC surgery was performed in 11 mice (groups I and II) to induce LV hypertrophy.
125
TAC is a commonly used procedure in mouse model for inducing cardiac hypertrophy 126
by increasing pressure overload, which is associated with a temporary enhancement 127
of the heart’s contractility [29]. For TAC surgery, anesthesia was induced using 4%
128
isoflurane and maintained with 2% isoflurane in an O2/N2 mixture with ratios 0.3/0.7, 129
respectively. Using a laboratory microscope, a ligation resulting in constriction of the 130
aorta was performed in the mouse via partial thoracotomy at the level of the second 131
rib. Thymus and fat tissue were separated from the aortic arch using forceps. A 7-0 132
silk suture was placed around the aorta between the brachiocephalic artery and the left 133
common carotid artery and tied around a 27-G needle. After ligation, the needle was 134
removed. The suture knots were tied slightly looser in the mild HCM mice (group I) 135
than in the severe HCM group (group II). The muscle and skin layers were closed 136
using 5-0 nylon sutures. Post-operative analgesia [carprofen (Rimadyl, Pfizer Oy 137
Animal Health, Helsinki, Finland) 5 mg/kg and buprenorphine (Temgesic - RB 138
Pharmaceuticals Limited, Slough, UK) 0.05-1.0 mg/kg] was administered on the day 139
of surgery and again at 2 days post-TAC. After surgery, sterile saline was 140
administered subcutaneously for hydration.
141
2.2. MRI 142
In vivo MR measurements were performed in a horizontal 9.4 T magnet equipped 143
with a Varian console (Agilent Inc., Santa Clara, California, USA). Inhalation 144
anesthesia during imaging was maintained with 1.2-1.5% isoflurane with a mixture of 145
30% O2 and 70% N2. A pad that circulated warm water was placed under the mouse 146
to keep the core temperature of the mouse close to 37°C. A volume transceiver RF 147
coil (Rapid Biomed GmbH, Germany) with 35 mm inner diameter was used for all 148
measurements. All mice were placed in the prone position, with their hearts 149
positioned at the isocenter of the magnet and RF coil. Electrocardiography (ECG) 150
and respirations were monitored using a small animal gating device (SA Instruments 151
Inc., NY, USA).
152
The slice selection for relaxation time measurements was performed based on ECG- 153
triggered and respiratory-gated gradient echo cine imaging (echo time (TE) = 1.9 ms, 154
repetition time (TR) = 4.6 ms, flip angle = 15°, 17-21 frames per cardiac cycle, field 155
of view = 30 x 30 mm2 and matrix size = 192 x 192). A short axis slice was selected 156
for the relaxation time measurements based on cine MRI in the mid line between apex 157
and base of the heart. Rotating frame relaxation times (T1ρ, TRAFF2 and TRAFF3) were 158
measured using an ECG- and respiratory-triggered TurboFLASH gradient echo 159
readout sequence (TE = 1.6 ms, FLASH TR = 3.1 ms, flip angle = 25°, data matrix = 160
128 x 128, Field-of-view = 30 x 30 mm2 and slice thickness = 1 mm). The TR 161
between the rotating frame weighting pulses was 2-3 s depending on the respiratory 162
cycle. T1ρ measurements were performed with a CW pulse power of 1250 Hz and 163
durations of 0, 18, 36, and 54 ms. A 90° flip of the magnetization and its return after a 164
CW pulse was accomplished using an adiabatic half-passage pulse (pulse length = 4 165
ms and RF power = 2500 Hz), as described previously [30]. For TRAFF2 and TRAFF3
166
measurements, the maximum RF pulse power was set to 1250 Hz, the length of the 167
composite pulse in the pulse train was 1.1 ms and the number of composite pulses in 168
the pulse train were 0, 16, 32, and 48; note that the pulses that were used were double 169
power and half duration compared to those used in the original RAFFn publication 170
[28]. The scanning time for T1ρ, TRAFF2 and TRAFF3 was 12-15 minutes each. The 171
overall imaging time for cine MRI, TRAFFn and CW T1ρ was 60-70 minutes, depending 172
on the heart and respiration rates of individual mice.
173
2.3. Data analysis 174
Maps from the signal intensities were reconstructed using Aedes (aedes.uef.fi) in the 175
MATLAB (Mathworks Inc., CA, USA) platform. The T1ρ andTRAFFn maps were fitted 176
using the linear regression model as described previously for RAFF maps [31]. The 177
fibrotic areas were identified in relaxation time maps based on the appearance of 178
fibrosis in histology sections. Regions of interest (ROI) were drawn in fibrotic and 179
remote muscle areas to measure relaxation times and calculate the relative relaxation 180
time difference (RRTD) from the relaxation time maps. RRTD = (T(fibrosis) - 181
T(Remote muscle)) / T(Remote muscle), where T is the average rotating frame 182
relaxation time (i.e., T1ρ, TRAFF2 andTRAFF3), which was calculated for all the 183
relaxation times and averaged over fibrotic or remote muscle areas. Collagen volume 184
fraction (CVF) was calculated from the histology by dividing the tissue area covered 185
by blue dye with total myocardial area. The results are presented as the means ± 186
standard deviation (SD). The statistical analysis was performed by one-way ANOVA, 187
t-test, two-way ANOVA and Bonferroni's post hoc tests.
188
2.4. Tissue processing and staining 189
All the animals were sacrificed for the histology after MR measurements were 190
performed. The hearts were rinsed with phosphate buffer saline (PBS) and 4%
191
paraformaldehyde in 7.5% sucrose was used for fixation. Paraformaldehyde was 192
replaced by 15% sucrose after 4 hours. Increasing concentrations of ethanol and 193
xylene were then used to dehydrate the hearts. The hearts were embedded in paraffin 194
blocks, and 4-µm sections were prepared from the blocks. The short axis slices were 195
cut, and a histological slice was selected based on the relaxation time measurements 196
by measuring the distance from apex to the mid line between apex and base of the 197
heart, i.e. to the location where both ventricles are visible. Tissue samples were then 198
deparaffinized and rehydrated with ethanol (100% - 50%), followed by Masson’s 199
trichrome staining. Images were acquired using a microscope (Nikon Eclipse, Ni-E, 200
Tokyo, Japan).
201
3. Results 202
MR in vivo measurements were performed in all the animals. RRTDs from the 203
fibrotic tissues and remote muscles were measured in mild and severe HCM groups (I 204
and II). Masson’s trichrome staining verified fibrosis in TAC-operated mice.
205
3.1. T
RAFFnin the myocardium
206
To our knowledge, this is the first report of TRAFFn in the myocardium in vivo (Tables 207
1 and 2). TRAFFn maps appeared to be artifact-free, and the overall relaxation times 208
were higher than T1ρ, whichwas expected (Figures 1 and 2). A small variations in 209
RRTDs of TRAFF3 (0.03 ± 0.02) and TRAFF2 (0.01±0.01) were found over the 210
myocardium in the intact hearts (III).
211
3.2.
RRTD of T1ρ increased in mild HCM group (I) 212The ROIs were drawn based on the histology images due to the smaller fibrotic areas 213
in the mild HCM group. Contrast differences in TRAFFn relaxation times between 214
fibrotic and remote myocardium were not clearly visible between fibrotic and remote 215
areas over several time points in the mild HCM group (Figure 1). A significant 216
increase in RRTD was observed in T1ρ between days 5 and 62 post-TAC in the mild 217
HCM group (p = 0.02, one-way ANOVA and Bonferroni's multiple comparisons test) 218
(Figure 3). The increasing trend in the RRTDs of TRAFF2 andTRAFF3 with the progress 219
of mild HCM was also significant during the 89-day follow-up period (p < 0.05, one- 220
way ANOVA and Bonferroni's multiple comparisons test). Longer overall relaxation 221
times in the myocardium were visualized in TRAFFn relaxation maps in comparison to 222
T1ρ (Figures 1 and 4). A small variation (0.01±0.01) of T1ρ RRTD was found over the 223
myocardium in the group (III).
224
3.3. TRAFFn and T1ρ increased in the severe HCM group (II) 225
In Figures 2 and 4, increased TRAFFn relaxation times can be clearly observed in the 226
fibrotic areas compared to the remote areas in the severe HCM group (II). Relaxation 227
times were significantly increased from day 5 to day 24 in TRAFF2 (p < 0.05, two-way 228
ANOVA and Bonferroni post hoc-tests) and TRAFF3 (p < 0.01, two-way ANOVA).
229
The overall TRAFF2 and TRAFF3 were significantly increased in group (II) compared to 230
group (I) (p < 0.01, two-way ANOVA). T1ρ was increased in severe HCM compared 231
to mild HCM at all time points (5, 10 and 24 days); however, the increasing trends 232
were not significantly different from group (I) (p = 0.10, two-way ANOVA) (Figure 233
3). The highest T1ρ RRTD value was observed in group (II) at 24 days post-TAC (i.e., 234
0.22 ± 0.02). Higher RRTDs were observed in TRAFFn compared to T1ρ, and the 235
RRTDs of TRAFF2 increased from 0.23 ± 0.08 at five days post-TAC to a maximum 236
value of 0.39 ± 0.07 at 24 days post-TAC in group (II).
237
3.4. Histological confirmation 238
Figure 5 shows myocardial fibrosis in hearts that were stained using Masson’s 239
trichrome. Fibrosis in group (I) mice appeared mostly around the large arteries at 89 240
days post-TAC (Figure 5(a)), while fibrosis was spread over the whole myocardium 241
in group (II) mice at 24 days post-TAC (Figure 5(b)). Figures 1 and 2 were co- 242
registered with histology images in figure 5 for the histological confirmation. Figure 6 243
shows significantly higher CVF percentage in the group (II) than in the group (I) (p <
244
0.001, unpaired t-test).
245
4. Discussion 246
MR rotating frame relaxation times were measured in TAC-operated mice at several 247
time points to characterize the progressing fibrosis in LV hypertrophy. In vivo 248
measurements of T1ρ, TRAFF2 and TRAFF3 showed higher overall RRTDs in the severe 249
HCM group (II) compared to the mild HCM group (I). Higher RRTD values indicated 250
a clear increase in myocardial fibrosis based on the Masson’s trichrome-stained 251
histology sections. The overall increase of TRAFFn relaxation times from day 5 to day 252
24 in the severe HCM group (II) visually correlated well with the progressive fibrotic 253
areas in the myocardium.
254
This study showed a significant increase of T1ρ in both the mild and severe HCM 255
groups (I and II) and a faster and more intense increase in the severe HCM group.
256
These results correlate well with the increased fibrosis visualized using histology. In a 257
previous study, a similar increase in T1ρ was found after 7 days of left anterior 258
descending artery occlusion in mice [32]. Together, these results indicate that T1ρ acts 259
as a general marker for fibrosis that is more widely applicable than for myocardial 260
infarcted tissue only.
261
Longer TRAFFn relaxation times in fibrotic tissue compared to T1ρ were observed in 262
both HCM groups (I and II). In group (II), TRAFF2 and TRAFF3 exhibited higher RRTDs 263
than in group (I). A significant difference was also found between the time points of 5 264
and 24 days post-TAC in the severe HCM group (II). An increase in the RRTDs of 265
TRAFF2 and TRAFF3 over time in both groups (I and II) most likely indicates progressive 266
fibrosis similar to that observed for T1ρ. The longer overall TRAFFn relaxation times in 267
group (II) than in group (I) at all time points may indicate that the RAFFn is more 268
sensitive to the molecular changes in the myocardium than T1ρ,which showed a small 269
difference between groups (I) and (II). Fibrosis results in scarring from the 270
accumulation of extracellular matrix proteins followed by the thickening of the 271
myocardium [33]. The increase in RRTDs of TRAFFn and T1ρ are most likely due to the 272
enlarged extracellular space caused by cell loss and fibrosis; however, alterations in 273
the pH or the macromolecular content and proton molecular exchange may contribute 274
to the increase as well [33-35]. In addition, the TRAFFn relaxation times generally 275
appeared to be longer than the on-resonance T1ρ [26].
276
The differences between the fibrotic areas in the mild (I) and severe (II) HCM groups 277
over time indicated progressive LV hypertrophy. The TAC mouse model was 278
optimized to achieve the desired LV hypertrophy using differences in the aortic 279
constriction, i.e., the pressure overload between the groups. The result of the pressure 280
overload difference was verified using histology (Figure 5). In this study, we 281
observed fibrosis using Masson’s trichrome staining at 24 days post-TAC in severe 282
(II) HCM hearts and at 89 days post-TAC in mild (I) HCM hearts. In group (I), 283
fibrosis began close to the larger arteries (Figure 5 (a)) and expanded to the overall 284
myocardium in group (II) (Figure 5 (b)). The Masson’s trichrome staining method 285
effectively showed the differences between mild and severe fibrosis of the 286
myocardium.
287
At the time points less than ten days from HCM coarctation, the inflammation may 288
occur and have an impact on increased relaxation times, especially on T2 at the 289
earliest time points. We found the lowest values of T2 relaxation times in the first time 290
points supporting an idea of negligible inflammation in the early time points.
291
However, this cannot be ruled out since our histology focused on later time points (24, 292
62 and 89 days in mild HCM and 24 days in severe HCM). A previous study in TAC 293
mice showed that the inflammatory pathways activation is a transient event in TAC 294
mouse model and induction of proinflammatory mediators are capable to suppress 295
acute inflammation in the early (3-7 day) time points [36].
296
Tissue relaxation during a CW pulse depends on RF power, difference between 297
delivered RF and local Larmor frequency Δω in the sample, and rotating frame 298
relaxation times T1ρ or T2ρ. In general, B1 >> γ -1Δω to fulfill spin-lock condition and 299
violation of the condition leads to imaging artifacts [37]. A way to overcome the 300
problem is to increase B1. However, it is not possible in many cases, but artifacts can 301
be minimized by using TRAFFn method with higher ‘n’ [28].
302
To keep the spins locked in the rotating frame experiments, a spin-lock field must be 303
greater than the local field gradients. For instance, on-resonance T1ρ results relatively 304
high SAR levels limiting the clinical use of T1ρ especially at a higher B0 and in the 305
body applications. Application of low RF power and short pulse durations decrease 306
SAR, but in some cases results limited contrast [38]. It has been shown that the higher 307
rotating frame contrast between white and grey matter with low SAR can be obtained 308
by applying TRAFFn in a rat and the human brains [28]. This study showed that the 309
similar conclusion can be drawn between remote and damaged tissue in the 310
myocardium using TRAFFn. 311
In conclusion, we showed that T1ρ, TRAFF2 andTRAFF3 can be used to monitor 312
progressive fibrosis in HCM. Additionally, T1ρ andTRAFFn can be mapped in the TAC 313
mouse model. Higher contrast differences between the fibrotic tissues and remote 314
areas were observed in severe TAC. Furthermore, the increase in TRAFFn with 315
increasing fibrosis compared to the CW T1ρ paves the way for TRAFFn to be used as a 316
marker for fibrosis in progressive HCM.
317
Conflicts of Interest 318
The authors declare that they have no conflicts of interest.
319
Funding 320
The authors thank the ‘Sigrid Juselius Foundation’ for the financial support.
321
Acknowledgments 322
The authors thank the following staff members for the help and support: Maarit 323
Pulkkinen, Alejandra Sierra Lopez and Jari Nissinen.
324
References 325
1. Roth GA, Forouzanfar MH, Moran AE, Barber R, Nguyen G, Feigin VL, 326
Naghavi M, Mensah GA, Murray CJL. Demographic and epidemiologic drivers 327
of global cardiovascular mortality. The New England Journal of Medicine.
328
2015;372:1333-1341.
329
2. Maron BJ. Hypertrophic cardiomyopathy: A systematic review." JAMA. 2002;
330
13;287(10):1308-20.
331
3. Hoey ETD, Teoh JK, Das I. Ganeshan A, Simpson H, Watkin RW. The emerging 332
role of cardiovascular MRI for risk stratification in hypertrophic Cardiomyopathy.
333
Clinical Radiology. 2014;69(3):221-30.
334
4. Maron BJ. Hypertrophic cardiomyopathy. Lancet. 1997; 350(9071):127-33.
335
5. Spirito P, Seidman CE, McKenna WJ, Maron BJ. Management of hypertrophic 336
cardiomyopathy. N Engl J Med. 1997;336 (11), 775-785.
337
6. Spirito P, Maron BJ, Bonow RO, Epstein SE. Severe functional limitation in 338
patients with hypertrophic cardiomyopathy and only mild localized left 339
ventricular hypertrophy. J Am Coll Cardiol. 1986;8(3):537-44.
340
7. Louie EK, Maron BJ. Hypertrophic cardiomyopathy with extreme increase in left 341
ventricular wall thickness. J Am Coll Cardiol. 1986;8(1):57-65.
342
8. Spirito P, Bellone P, Harris KM, Bernabò P, Bruzzi P, Maron BJ. Magnitude of 343
left ventricular hypertrophy predicts the risk of sudden death in hypertrophic 344
cardiomyopathy. N Engl J Med. 2000;342(24):1778-85.
345
9. Elliott PM, Gimeno Blanes JR, Mahon NG, Poloniecki JD, McKenna WJ.
346
Relation between severity of left-ventricular hypertrophy and prognosis in 347
patients with hypertrophic cardiomyopathy. Lancet. 2001;357(9254):420-4.
348
10. Elliott PM, Poloniecki J, Dickie S, Sharma S, Monserrat L, Varnava A, Mahon 349
NG, McKenna WJ. Sudden death in hypertrophic cardiomyopathy: identification 350
of high risk patients. J Am Coll Cardiol. 2000;36(7):2212-8.
351
11. Allison JD, Flickinger FW, Wright JC, Falls DG 3rd, Prisant LM, VonDohlen 352
TW, Frank MJ. Measurement of left ventricular mass in hypertrophic 353
cardiomyopathy using MRI: comparison with echocardiography. Magn Reson 354
Imaging. 1993;11(3):329-34.
355
12. Posma JL, Blanksma PK, van der Wall EE, Hamer HP, Mooyaart EL, Lie KI.
356
Assessment of quantitative hypertrophy scores in hypertrophic cardiomyopathy:
357
magnetic resonance imaging versus echocardiography. Am Heart J.
358
1996;132(5):1020-‐7.
359
13. Higgins CB, Byrd BF 3rd, Stark D, McNamara M, Lanzer P, Lipton MJ, Schiller 360
NB, Botvinick E, Chatterjee K. Magnetic resonance imaging in hypertrophic 361
cardiomyopathy. Am J Cardiol. 1985;55(9),1121–1126.
362
14. Park JH, Kim YM, Chung JW, Park YB, Han JK, Han MC. MR imaging of 363
hypertrophic cardiomyopathy. Radiology. 1992;185(2),441-446.
364
15. Sipola, P, Lauerma K, Jääskeläinen P, Laakso M, Peuhkurinen K, Manninen H, 365
Aronen HJ, Kuusisto J. Cine MR imaging of myocardial contractile impairment 366
in patients with hypertrophic cardiomyopathy attributable to Asp175Asn 367
mutation in the alpha -tropomyosin gene. Radiology. 2005;236(3),815-824.
368
16. Doltra A, Amundsen BH, Gebker R, Fleck E, Kelle S. Emerging concepts for 369
myocardial late gadolinium enhancement MRI. Current Cardiology Reviews.
370
2013;9(3):185-90.
371
17. Amano Y, Tachi M, Tani H, Mizuno K, Kobayashi Y, Kumita S. T2-weighted 372
cardiac magnetic resonance imaging of edema in myocardial diseases. Scientific 373
World Journal. 2012;2012:194069.
374
18. Ferreira VM, Piechnik SK, Dall'Armellina E, Karamitsos TD, Francis JM, 375
Choudhury RP, Friedrich MG, Robson MD, Neubauer S. Non-contrast T1- 376
mapping detects acute myocardial edema with high diagnostic accuracy: a 377
comparison to T2-weighted cardiovascular magnetic resonance. Journal of 378
Cardiovascular Magnetic Resonance. 2012;14:42.
379
19. Hinojar R, Varma N, Child N, Goodman B, Jabbour A, Yu CY, Gebker R, Doltra 380
A, Kelle S, Khan S, Rogers T, Arroyo Ucar E, Cummins C, Carr-White G, Nagel 381
E, Puntmann VO. T1 mapping in discrimination of hypertrophic phenotypes:
382
hypertensive heart disease and hypertrophic cardiomyopathy findings from the 383
international T1 multicenter cardiovascular magnetic resonance study.
384
Circulation-Cardiovascular Imaging. 2015; 8(12). pii: e003285.
385
20. Flett AS, Hasleton J, Cook C, Hausenloy D, Quarta G, Ariti C, Muthurangu V, 386
Moon JC. Evaluation of techniques for the quantification of myocardial scar of 387
differing etiology using cardiac magnetic resonance. J Am Coll Cardiol 388
Cardiovasc Imaging. 2011;4(2):150-6.
389
21. Flett AS, Hayward MP, Ashworth MT, Hansen MS, Taylor AM, Elliott PM, 390
McGregor C, Moon JC. Equilibrium contrast cardiovascular magnetic resonance 391
for the measurement of diffuse myocardial fibrosis: preliminary validation in 392
humans. Circulation. 2010;122(2):138-44.
393
22. Puntmann VO, Voigt T, Chen Z, Mayr M, Karim R, Rhode K, Pastor A, Carr- 394
White G, Razavi R, Schaeffter T, Nagel E. Native T1 mapping in differentiation 395
of normal myocardium from diffuse disease in hypertrophic and dilated 396
cardiomyopathy. JACC: Cardiovascular Imaging. 2013;6(4):475-84.
397
23. Witschey WRT, Pilla JJ, Ferrari G, Koomalsingh K, Haris M, Hinmon R, Zsido 398
G, Gorman JH 3rd, Gorman RC, Reddy R. Rotating frame spin lattice relaxation 399
in a swine model of chronic, left ventricular myocardial infarction. Magnetic 400
Resonance in Medicine. 2010;64(5):1453-60.
401
24. Berisha S, Han J, Shahid M, Han Y, Witschey WRT. Measurement of myocardial 402
T1ρ with a motion corrected, parametric mapping sequence in humans. PLoS 403
ONE. 2016;11(3):e0151144.
404
25. Kettunen MI, Gröhn OH, Penttonen M, Kauppinen RA. Cerebral T1ρ relaxation 405
time increases immediately upon global ischemia in the rat independently of 406
blood glucose and anoxic depolarization. Magnetic Resonance in Medicine.
407
2001; 46(3):565-72.
408
26. Liimatainen T, Sorce DJ, O'Connell R, Garwood M, Michaeli S. MRI contrast 409
from relaxation along a fictitious field (RAFF). Magnetic Resonance in 410
Medicine. 2010;64(4):983-94.
411
27. Liimatainen T, Mangia S, Ling W, Ellermann J, Sorce DJ, Garwood M, Michaeli 412
S. Relaxation dispersion in MRI induced by fictitious magnetic fields. Journal of 413
Magnetic Resonance. 2011;209(2):269-76.
414
28. Liimatainen T, Hakkarainen H, Mangia S, Huttunen JMJ, Storino C, Idiyatullin D, 415
Sorce D, Garwood M, Michaeli S. MRI contrasts in high rank rotating frames.
416
Magn Reson Med. 2015;73(1):254-62.
417
29. DeAlmeida AC, Van Oort RJ, Wehrens XHT. Transverse aortic constriction in 418
mice. Journal of Visualized Experiments. 2010;(38). pii:1729.
419
30. Gröhn OHJ, Kettunen MI, Mäkelä HI, Penttonen M, Pitkänen A, Lukkarinen JA, 420
Kauppinen RA. Early detection of irreversible cerebral ischemia in the rat using 421
dispersion of the magnetic resonance imaging relaxation time, T1rho. J Cereb 422
Blood Flow Metab. 2000;20(10):1457-66.
423
31. Liimatainen T, Sierra A, Hanson T, Sorce DJ, Ylä-Herttuala S, Garwood M, 424
Michaeli S, Gröhn O. Glioma cell density in a rat gene therapy model gauged by 425
water relaxation rate along a fictitious magnetic field. Magn Reson Med.
426
2012;67(1):269-77.
427
32. Musthafa H-S N, Dragneva G, Lottonen L, Merentie M, Petrov L, Heikura T, 428
Ylä-Herttuala E, Ylä-Herttuala S, Gröhn O, Liimatainen T. Longitudinal Rotating 429
Frame Relaxation Time Measurements in Infarcted Mouse Myocardium in Vivo.
430
Magnetic Resonance in Medicine. 2013;69(5):1389-95.
431
33. Neary R, Watson CJ, Baugh JA. Epigenetics and the Overhealing Wound: The 432
Role of DNA Methylation in Fibrosis. Fibrogenesis & Tissue Repair. 2015;8:18.
433
34. Michaeli S, Sorce DJ, Idiyatullin D, Ugurbil K, Garwood M. Transverse 434
relaxation in the rotating frame induced by chemical exchange. J Magn Reson.
435
2004;169(2):293-9.
436
35. Michaeli S, Sorce DJ, Springer CS Jr, Ugurbil K, Garwood M. T1rho MRI 437
contrast in the human brain: modulation of the longitudinal rotating frame 438
relaxation shutter-speed during an adiabatic RF pulse. J Magn Reson.
439
2006;181(1):135-47.
440
36. Xia Y, Lee K, Li N, Corbett D, Mendoza L, Frangogiannis NG. Characterization 441
of the Inflammatory and Fibrotic Response in a Mouse Model of Cardiac 442
Pressure Overload. Histochem Cell Biol. 2009; 131(4): 471–481.
443
37. Witschey WR, Borthakur A, Elliott MA, Mellon E, Niyogi S, Wallman DJ, Wang 444
C, Reddy R. Artifacts in T1 rho-weighted imaging: compensation for B(1) and 445
B(0) field imperfections. J Magn Reson. 2007;186(1):75-85.
446
38. Witschey WR, Zsido GA, Koomalsingh K, Kondo N, Minakawa M, Shuto T, 447
McGarvey JR, Levack MM, Contijoch F, Pilla JJ, Gorman JH 3rd, Gorman RC.
448
In vivo chronic myocardial infarction characterization by spin locked 449
cardiovascular magnetic resonance. J Cardiovasc Magn Reson. 2012;14:37.
450 451
Table 1: In vivo relaxation times and RRTDs (Means ± SD) of fibrotic myocardium 452
and chest muscle areas (n=6) for the mild HCM group (I) 453
TRAFF2 5 10
Day
24 62 89
Myocardium [ms] 52.00±6.00 57.20±6.01 54.00±4.42 57.00±5.89 63.00±8.71 Chest muscle [ms] 46.20±3.63 47.40±2.40 46.50±3.32 47.67±3.14 48.67±3.78 RRTD 0.12±0.04 0.20±0.08 0.16±0.06 0.19±0.07 0.29±0.08
TRAFF3 5 10 24 62 89
Myocardium [ms] 75.80±8.13 83.80±7.19 81.00±6.41 82.67±8.61 96.00±16.52 Chest muscle [ms] 67.40±5.41 69.20±3.19 67.33±7.14 69.00±5.06 74.67±9.29 RRTD 0.12±0.03 0.20±0.05 0.20±0.07 0.19±0.08 0.28±0.06
T1ρ 5 10 24 62 89
Myocardium [ms] 27.00±1.00 27.60±2.40 28.17±2.22 29.33±1.03 29.00±2.00 Chest muscle [ms] 25.20±0.83 24.20±1.09 23.67±1.36 24.17±0.75 24.00±1.73 RRTD 0.07±0.01 0.14±0.10 0.19±0.08 0.21±0.05 0.20±0.04 454
Table 2: In vivo relaxation times and RRTDs (Means ± SD) of fibrotic myocardium 455
and chest muscle areas (n=5) for the severe HCM group (II).
456 457
Day
TRAFF2 5 10 24
Myocardium [ms] 60.66±10.69 63.50±3.87 68.00±13.61 Chest muscle [ms] 49.00±5.19 49.75±2.36 48.75±7.22
RRTD 0.23±0.08 0.27±0.06 0.39±0.07
TRAFF3 5 10 24
Myocardium [ms] 87.33±15.50 94.75±4.57 100.25±15.12 Chest muscle [ms] 73.33±7.57 72.25±4.11 73.25±9.74
RRTD 0.18±0.09 0.31±0.10 0.36±0.06
T1ρ 5 10 24
Myocardium [ms] 28.33±2.30 28.50±0.57 28.00±1.82 Chest muscle [ms] 24.66±2.08 24.50±0.57 23.00±1.82
RRTD 0.15±0.02 0.16±0.03 0.22±0.02
458 459
Figure legends 460
Figure 1: In vivo rotating frame relaxation time maps of mice of mild HCM group (I) 461
at 5, 10, 24, 62 and 89 days post-TAC.
462 463
Figure 2: In vivo rotating frame relaxation time maps of mice of severe HCM group 464
(II) at 5, 10 and 24 days post-TAC. Overall relaxation times were increased from day 465
5 to day 24 post-TAC in TRAFF2 andTRAFF3. 466
467
Figure 3: Time evolution of the RRTD values (Means ± SD) (a) T1ρ and (b) TRAFF2
468
and TRAFF3 in the mild HCM group (I) and in the severe HCM group (II). *P<0.05 and 469
**P< 0.01, one-way ANOVA, two-way ANOVA, Bonferroni's post hoc tests.
470 471
Figure 4: In vivo rotating frame relaxation time maps of control (intact heart), mild 472
HCM (after 89 days post-TAC) and severe HCM mice (after 24 days post-TAC).
473 474
Figure 5: Masson’s trichrome stain (a) at 89 days post-TAC in the representative 475
mild group (I) and (b) at 24 days post-TAC in the severe HCM group (II) mouse heart.
476
In both images, the fibrosis is observed around the large arteries (white arrows) and 477
the larger fibrotic areas (black arrows).
478 479
Figure 6: Collagen volume fraction (CVF) (Mean ± SD) measurements from 480
Masson’s trichrome stained histology images of mild and severe groups (I and II).
481
***P < 0.001, unpaired t-test.
482 483
