1. In Vivo Analysis of Troponin C Knock-In (A8V) MiceCLINICAL PERSPECTIVE
by Adriano S. Martins, Michelle S. Parvatiyar, Han-Zhong Feng, J. Martijn Bos, David Gonzalez-Martinez, Milica Vukmirovic, Rajdeep S. Turna, Marcos A. Sanchez-Gonzalez, Crystal-Dawn Badger, Diego A.R. Zorio, Rakesh K. Singh, Yingcai Wang, J.-P. Jin, Michael J. Ackerman, and Jose R. Pinto
Circ Genom Precis Med
Volume 8(5):
October 20, 2015
Copyright © American Heart Association, Inc. All rights reserved.

2. Echo- and electro-cardiograms of the TNNC1-A8V patient.
Echo- and electro-cardiograms of the TNNC1-A8V patient. A, ECHO image from apical 4-chamber showing mild to moderate left atrial enlargement (white arrow). B, ECHO image from parasternal long axis showing basal to midanteroseptal and posterior wall hypertrophy (white arrows) as an indication of concentric left ventricle hypertrophy. C–G, ECG tracing from patient showing sinus rhythm, pathological Q-waves suggesting prior ischemia or hypertrophic cardiomyopathy (C–F), increased QRS amplitude suggesting left ventricular hypertrophy (E and G). Ao indicates aorta; LA, left atria; LV, left ventricle; and RV, right ventricle.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

3. Echocardiography and ex vivo working heart function of the KI-TnC-A8V mice.
Echocardiography and ex vivo working heart function of the KI-TnC-A8V mice. A representative ECHO image of the left ventricle is shown in A from a 14 m wild-type (WT) mouse and in B from a 14 m KI-TnC-A8V+/+ mouse. The response to β-adrenergic stimulation in ex vivo working heart function in 12 to 13 m mice is shown in C. Stroke volume normalized to the heart weight; D, LVPmax (maximal left ventricular pressure); E, LVPmin (minimal left ventricular pressure); F, +dP/dt (rate of rise of left ventricular pressure); G, −dP/dt (rate of fall of left ventricular pressure). Data are shown as mean±SE, n=5 mice. **P<0.01 and ***P<0.001 KI vs WT; ##P<0.01 and ###P<0.001 KI-TnC-A8V+/+ vs KI-TnC-A8V+/− using 2-way ANOVA with Tukey adjustment. &P<0.05 KI-TnC-A8V+/+ vs KI-TnC-A8V+/−, and $P<0.05 KI-TnC-A8V+/+ vs WT and KI-TnC-A8V+/− without ISO treatment using 1-way ANOVA with Tukey adjustment. ISO indicates isoproterenol.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

4. Heart weight and hypertrophic cardiomyopathy gene expression profile in KI-TnC-A8V mice.
Heart weight and hypertrophic cardiomyopathy gene expression profile in KI-TnC-A8V mice. The body weight (A); the ratio heart weight/body weight (B); the left atria weight (C); and the ratio left atria/heart weight (D). A–D are data from 12 to 13 m mice. Data are shown as mean±SE, n=5 mice. *P<0.05, **P<0.01, and *** P<0.001 KI vs WT; ##P<0.01 and ###P<0.001 KI-TnC-A8V+/+ vs KI-TnC-A8V+/−. E and F, The mRNA expression levels of cardiac hypertrophic markers in 16 to 18 m mice. Data are shown as mean±SE, n=6 mice. *P<0.05 KI vs WT; #P<0.05 KI-TnC-A8V+/+ vs KI-TnC-A8V+/−. ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; and MHCβ, myosin heavy chain isoform β.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

5. Histopathologic analysis of KI-TnC-A8V mouse hearts at 16 to 18 months of age.
Histopathologic analysis of KI-TnC-A8V mouse hearts at 16 to 18 months of age. Representative Masson trichrome stained sections were imaged at 10× magnification. A–C, The black arrows indicate the location of papillary muscles. Papillary muscle hypertrophy can be seen in KI-TnC-A8V+/− and KI-TnC-A8V+/+ mouse hearts. Evidence of papillary muscle hypertrophy can also be seen at lower magnification in Figure IIA–IIC in the Data Supplement. E–F, Interstitial fibrosis can be seen in heart sections obtained from the same group of mice. G, The retention time of the cTnC peptides separated by liquid chromatography mass spectrometry. The graphs show the ratio cTnC A8V:WT in the KI-TnC-A8V+/− hearts quantified by the abundance of 15 and 11 amino acid peptides. n=3.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

6. Sarcomere length (SL) and intracellular Ca2+ levels in KI-TnC-A8V intact cardiomyocytes at different frequencies of stimulation.
Sarcomere length (SL) and intracellular Ca2+ levels in KI-TnC-A8V intact cardiomyocytes at different frequencies of stimulation. A, SL is measured under resting conditions; B, Percentage of SL shortening; C, Diastolic intracellular Ca2+ levels; D, Ca2+ amplitude is represented as the percentage of increase above diastolic intracellular Ca2+ levels in cardiomyocytes obtained from 3-mo-old mice. Data are shown as mean±SE, n=3 to 4 mice. *P<0.05 KI vs WT; #P<0.05, KI-TnC-A8V+/+ vs KI-TnC-A8V+/− at the same frequency of stimulation. The values for each point are presented in Table I in the Data Supplement.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

7. Kinetics of contractility and Ca2+ transients in KI-TnC-A8V intact cardiomyocytes electrically stimulated at 4 and 6 Hz. A, Time for the sarcomere length (SL) to return to 50% of baseline (RT, relaxation time); the rate of cell relengthening (−dL/dt); and time for SL shortening to achieve the peak value.
Kinetics of contractility and Ca2+ transients in KI-TnC-A8V intact cardiomyocytes electrically stimulated at 4 and 6 Hz. A, Time for the sarcomere length (SL) to return to 50% of baseline (RT, relaxation time); the rate of cell relengthening (−dL/dt); and time for SL shortening to achieve the peak value. B, Time for intracellular Ca2+ to return to 50% of baseline and the time for intracellular Ca2+ to achieve peak during systole. Data are shown as mean±SE, n=3 to 4 mice. *P<0.05 KI vs WT; #P<0.05, KI-TnC-A8V+/+ vs KI-TnC-A8V+/−. The values for each point are presented in Table II in the Data Supplement.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

8. The Ca2+ sensitivity of force development and rate of relaxation in papillary skinned fibers and immunoblots of Ca2+-handling proteins in KI-TnC-A8V hearts.
The Ca2+ sensitivity of force development and rate of relaxation in papillary skinned fibers and immunoblots of Ca2+-handling proteins in KI-TnC-A8V hearts. A, The Ca2+ dependence of force generation in cardiac skinned fibers from 4-mo-old mice. Data are shown as mean±SE, n=9 to 11. *P<0.05 KI vs WT; #P<0.05 KI-TnC-A8V+/+ vs KI-TnC-A8V+/−. B, A representative tracing measuring the rate of relaxation in cardiac skinned fibers investigated by Diazo-2 on flash photolysis. The amplitudes (A, %) and rate constants (k, s−1) obtained from 4-mo-old mice were as follows; WT: A1=83.82±2.06, k1=15.48±0.9, A2=14.56±2.47, and k2=2.01±0.53; KI-TnC-A8V+/+: A1=84.24±5.06, k1=9.42±0.84, A2=14.28±4.36, and k2=0.75±0.18. k1 and k2 are statistically different between WT and KI-TnC-A8V+/+ (P<0.05). The t1/2 (ms) of skinned fiber relaxation was also statistically different between WT (55.62±4.25) and KI-TnC-A8V+/+ (82.86±6.78), n=8 to 11. C, Representative immunoblots for the detection of SERCA2, PLN-T, phosphorylated PLN at serine 16 (PLN-PSer16), NCX1, CASQ2 and phosphorylated cTnI at serines 23, 24 (cTnI-P) in the left ventricle of 9 mo-old WT, KI-TnC-A8V+/− (+/−), and KI-TnC-A8V+/+ (+/+) mice. For all experiments, tubulin was used as an internal control. D, Quantification of the protein expression and phosphorylation levels. Data are shown as mean±SE, n=6 mice. *P<0.05 KI vs WT; #P<0.05 KI-TnC-A8V+/+ vs KI-TnC-A8V+/−.
Adriano S. Martins et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.