Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model

Table 1 Details of cardiac specific gene primers

No.	Gene primers	Primer sequences (5׳-3׳)	Product size
1	GAPDH (F)	GGAAAGCTGTGGCGTGATGG	414
	GAPDH (R)	GTAGGCCATGAGGTCCACCA
2	GATA4 (F)	TCTCACTATGGGCACAGCAG	245
	GATA4 (R)	CCGAGCAGGAATTTGAAGAG
3	cTnT (F)	TTCGACCTGCAGGAAAAGTT	206
	cTnT (R)	GTGCCTGGCAAGACCTAGAG
4	Nkx2.5 (F)	ACCGCCCCTACATTTTATCC	230
	Nkx2.5 (R)	GACAGGTACCGCTGTTGCTT
5	Connexin43 (F)	GCTCCACTCTCGCCTATGTC	234
	Connexin43 (R)	GAGTTCATGTCCAGCAGCAA
6	cMHC (F)	GCAGGTGGATGATCTGGAGG	116
	cMHC (R)	CCATGATGCTCTCCTGGGTC
7	Tbx20 (F)	TGATTCTGGCCCTAGGAAAGTG	100
	Tbx20 (R)	TGAGCCTGGGTTCTCCATAAAG
8	ANF (F)	CCTGCGAAGATCAAGCTGC	110
	ANF (R)	GCTCGAGCAGATTTGGCTG
9	cTnI (F)	GACCTGCGTGGCAAGTTTA	139
	cTnI (R)	CCTCCTTCTTCACCTGCTTG
10	Mef2c (F)	GAGATACCCACAACACACGC	117
	Mef2c (R)	GAGTGGAATTCGTTCCGGTG

F: Forward; R: Reverse; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; cTnT: Cardiac troponin T; cMHC: Cardiac myosin heavy chain; Tbx20: T-box transcription factor 20; ANF: Atrial natriuretic factor; cTnI: Cardiac troponin I; Mef2C: Myocyte-specific enhancer-binding factor 2C.

Table 2 Degrees of freedom (in vitro experiments)

Experiment	Degrees of freedom (F)
MTT assay	F3,8 = 404.312
Gene expression
GATA4	F3,8 = 14.883
cTnT	F3,8 = 15.870
cTnI	F3,8 = 34.024
Nkx2.5	F3,8 = 15.901
cMHC	F3,8 = 85.276
Connexin43	F3,8 = 19.912
ANF	F3,8 = 7.510
Tbx20	F3,8 = 22.877
Mef2C	F3,8 = 7.660
Protein expression
Percent positive cells
GATA4	F3,8 = 26.609
cTnT	F3,8 = 46.829
Nkx2.5	F3,8 = 513.556
Fluorescence intensity
GATA4	F3,8 = 9.696
cTnT	F3,8 = 28.438
Nkx2.5	F3,12 = 59.175

cTnT: Cardiac troponin T; cTnI: Cardiac troponin I; cMHC: Cardiac myosin heavy chain; ANF: Atrial natriuretic factor; Tbx20: T-box transcription factor 20; Mef2C; Myocyte-specific enhancer-binding factor 2C.

Table 3 Echocardiographic parameters

Groups	Duration	LVIDd (cm)	LVIDs (cm)	EF%	FS%	EDV (mL)	ESV (mL)	SV (mL)
Sham control	2 wk	0.4194	0.1639	93.5889	60.9556	0.0722	0	0.0667
MI + ve		0.5156	0.3722	62.5556	28.3056	0.1389	0.05	0.1056
MI + scaffold		0.5193	0.332	74.4467	37.3067	0.1533	0.04	0.1133
MI + MSC-seeded scaffold		0.4461	0.2611	78.8	40.9389	0.1167	0	0.0667
MI + zebularine treated MSC-seeded scaffold		0.5144	0.2972	80.2111	42.1278	0.1444	0	0.1278
Sham control	4 wk	0.4705	0.195	92.7389	59.2222	0.1222	0	0.1111
MI + ve		0.5780	0.4363	57.3306	24.9194	0.1889	0.0833	0.1222
MI + scaffold		0.47	0.305	73.1056	36.3278	0.1222	0.0333	0.0833
MI + MSC-seeded scaffold		0.5772	0.3455	78.0056	40.5667	0.2111	0.0389	0.1667
MI + zebularine treated MSC-seeded scaffold		0.5338	0.285	82.6056	45.6611	0.1667	0	0.1222
Sham control	6 wk	0.5183	0.2333	90.3333	54.8667	0.15	0	0.1333
MI + ve		0.7066	0.5716	46.85	19.55	0.3444	0.2	0.1611
MI + scaffold		0.5472	0.3622	70.0444	33.8889	0.1667	0.0389	0.1278
MI + MSC-seeded scaffold		0.5533	0.3288	78.05	40.8611	0.1722	0.0333	0.1444
MI + zebularine treated MSC-seeded scaffold		0.5589	0.3156	81.7028	43.7972	0.17778	0.0167	0.1528

LVIDd: Left ventricular internal diameter diastole; LVIDs: Left ventricular internal diameter systole; EF: Ejection fraction; FS: Fractional shortening; EDV: End diastolic volume; ESV: End systolic volume; SV: Stroke volume; MI: Myocardial infarction; MSC: Mesenchymal stem cells.

Table 4 Degrees of freedom (in vivo experiments)

Experiments		Degrees of freedom (F)
Cardiac function analysis	Parameters	Duration, 2 wk		Duration, 4 wk	Duration, 6 wk
	LVIDd	F4,25 = 1.936		F4,25 = 1.299	F4,25 = 3.594
	LVIDs	F4,25 = 9.060		F4,25 = 6.944	F4,25 = 14.008
	%EF	F4,25 = 26.475		F4,25 = 18.378	F4,25 = 24.089
	%FS	F4,25 = 35.299		F4,25 = 24.143	F4,25 = 26.754
	EDV	F4,25 = 1.571		F4,25 = 1.059	F4,25 = 3.601
	ESV	F4,25 = 3.44		F4,25 = 5.339	F4,25 = 9.580
	SV	F4,25 = 2.305		F4,25 = 0.477	F4,25 = 0.262
Immunohistochemistry	α-SMA	F4,10 = 31.551
Cell tracking	Fluorescence intensities of cardiac specific proteins
	cMHC		F = 1.631
	cTnI		F = 0.882
	cTnT		F = 2.665
	Fluorescence intensities of DiI labeled cells
	cMHC		F = 0.398
	cTnI		F = 0.089
	cTnT		F = 0.039

LVIDd: Left ventricular internal diameter diastole; LVIDs: Left ventricular internal diameter systole; EF: Ejection fraction; FS: Fractional shortening; EDV: End diastolic volume; ESV: End systolic volume; SV: Stroke volume; α-SMA: α-smooth muscle actin; cMHC: Cardiac myosin heavy chain; cTnI: Cardiac troponin I; cTnT: Cardiac troponin T.