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Online Appendix for the following JACC article

TITLE: Elevated Pre-Operative Serum Peptides for Collagen I and III Synthesis Result in Post-Surgical Atrial Fibrillation

AUTHORS: Michael F. Swartz, PhD, Gregory W. Fink, MD, Muhammad F. Sarwar, MD, George L. Hicks, MD, Yao Yu, PhD, Rui Hu, PhD, Charles J. Lutz, MD, Steven M. Taffet, PhD, José Jalife, MD

APPENDIX

Data Supplement for

Elevated Serum Peptides for Collagen I and III Synthesis Result in Post-Surgical Atrial Fibrillation

Methods

Echocardiographic Measurements

Pre-operative echocardiograms were obtained in all patients. Two-dimensional imaging, pulsed, and color Doppler recordings were performed; left ventricular mass index was calculated after measuring the septal and posterior ventricular wall thickness and dividing the ventricular mass by the body surface area. The pulsed Doppler measurements obtained were: maximal early transmitral velocity in diastole (E wave); maximal late transmitral velocity in diastole (A wave); A-wave duration; and mitral annular movements during the E and A waves (E’ wave and A’ wave). The left ventricular ejection fraction was determined and LA and right atrial (RA) diameters measured from a 4-chamber view during atrial diastole.

RNA Methods

RNA was extracted using a Qiagen RNeasy minikit, and transcribed using an Invitrogen Superscript III first strand synthesis kit. Primers (Table 1 supplement) were selected to cross an intron within the DNA sequence of interest. Twenty-five µl reactions using SyberGreenERTM (Invitrogen) were run in duplicate on a BioRad iCycler 96 well plate. The cycles to threshold (cT) values corresponding to messenger RNA (mRNA) levels are based on a log scale and were transformed to delta cT values by subtracting the gene of interest from (18s rRNA). 18s rRNA expression is present in all cells, and accounts for variability in RNA quality. To compare LA versus RA differences, the respective data were transformed from cT values to equivalent fold differences, by using the following equation:

Fold difference (mean ΔcTRA_AF - mean ΔcTLA_AF) = 2(mean ΔcTRA_AF - mean ΔcTLA_AF).

The majority of AF occurred on post-operative day number 2 (Table 2 Supplement). The mean duration of AF was 3.2 ±2.8 hours. Ten patients had greater than 1 episode of AF, and two patients were discharged from the hospital in AF.

Statistical Methods

Patient demographics and echocardiogram information are reported as mean ± standard deviation or n (%). Patient variables and left to right atrial comparisons were evaluated for homogeneity of variance, as well as normality, and comparisons made using a 2-tailed Student’s t-test for the independent and paired samples, respectively. Comparisons between the categorical variables were carried out using Pearson’s chi square or Fisher’s exact test, as appropriate to the data. Differences between atrial biopsies in patients who developed AF or remained in NSR were compared using 2-way ANOVA with 1 repeated measures factor (left vs right). Serum sample data were compared using an ANOVA model with a post-hoc Bonferroni adjustment. Receiver operating characteristic (ROC) curves were constructed for serum sample values between patients who developed AF and those who remained in NSR. The percentage area of the graph under the ROC curve (AUC) was used to validate statistical significance. To compare age and serum markers, a binary logistic regression was performed regressing age on serum markers, and the AUC for the model was determined. A multivariate logistic regression model was used to test the effects of covariates upon the outcome of AF using a backward procedure to select the final model according to the Akaike information criterion. The covariates selected by this procedure demonstrated the best model among all candidate models with different combinations of the covariates to predict AF. A linear model was used to evaluate the relationship between the percentage of fibrosis and the serum levels of each peptide and that relationship is summarized by the R2 and its associated p-value.

Table 1. Forward and Reverse Primer Sequences

|Gene |Sequence |Base Pairs |

|18S rRNA |CCCGAAGCGTTTAACTTTGAA |136 |

| |CCCTCTTAATCATGGCCTCA | |

|COL-I |GCTACCCAACTTGCCTTCATG |33 |

| |GCTGTTCTTGCAGTGGTAGGTG | |

|COL-III |CCCACTATTATTTTGGCACAACAG |56 |

| |GCATGGTTCTGGCTTCCAGA | |

|TGF-β |CCAGCATCTGCAAAGCTCC |94 |

| |GGTCCTTGCGGAAGTCAATGT | |

|PDGF |ACACGAGCAGTGTCAAGTGC |180 |

| |GGCTCATCCTCACCTCACAT | |

|Angiotensin |GAACTGGATGTTGCTGCTGA |111 |

| |GGAGAAGCCCTTCATCTTCC | |

|TIMP-1 |AATTCCGACCTCGTCATCAG |229 |

| |TGCAGTTTTCCAGCAATGAG | |

|MMP-1 |CATGCGCACAAATCCCTTCT |110 |

| |CATCTCTGTCGGCAAATTCGT | |

|MMP-9 |TTGACAGCGACAAGAAGTGG |178 |

| |GCCATTCACGTCGTCCTTAT | |

Abbreviations: COL-Collagen, 18s rRNA-Ribosomal RNA 18s, PDGF-Platelet Derived Growth Factor, TGFβ-Transforming Growth Factor Beta

Table 2. AF Characteristics

Day of Development

POD 1 2

POD 2 8

POD 3 5

POD 4 2

POD 5 1

Duration of AF (hours) 3.19 ± 2.8

Episodes of AF 1.6 ± 0.77

1 8

2 6

3 ; 4

Discharged in AF 2

Abbreviations: POD-Post Operative Day, AF-Atrial Fibrillation

Table 3 Variables for Patients with Atrial Biopsies

NSR = 33 AF after Surgery = 11 P value

Age (years) 63.6±8.2 74.4±7.0 0.001*

Male Gender 27 (82%) 9 (82 %) 1.0

BSA m2 2.08±0.2 2.02±0.2 0.4

Medical History

Hypertension 27 (82%) 9 (82%) 1.0

COPD 3 (9%) 0 (0%) 0.5

Hyperthyroidism 0 0 1.0

CAD 33 (100%) 11 (100%) 1.0

Tobacco Abuse 19 (57%) 6 (54%) 0.8

Alcohol Abuse 1 (3%) 0 1.0

Pre-operative Medications

ACE Inhibitor 12(36%) 3(27%) 0.8

ARB 5 (15%) 0 (0%) 0.7

Beta Blocker 15 (45%) 6 (54%) 0.2

Statin 20 (60%) 4 (36%) 0.2

P-Wave Duration (sec) 0.12±0.01 0.12±0.01 0.9

Operation

CABG 32 (97%) 10(91%) 0.5

Valve 0 (0%) 0 (0%) 1.0

CABG/Valve 1 (3%) 1 (9%) 0.4

Abbreviations: AF-Atrial Fibrillation; ACE, Angiotensin Converting Enzyme inhibitors; BSA, Body surface area; ARB, Angiotensin Receptor Blockers; CABG-Coronary Artery Bypass Graft; COPD, Chronic Obstructive Pulmonary Disease; HTN-Hypertension; NSR-Normal Sinus Rhythm; NYS CHF- New York State Congestive Heart Failure; Valve, Valvular Heart Disease.

*Denotes Statistical Significance

Table 4 18s rRNA Expression

|Gene |LA |RA |p value |

|18s rRNA |23.2 ± 0.3 |23.7 ± 0.6 |0.2 |

|Gene |LA-AF |LA-NSR |p value |RA-AF |RA-NSR |p value |

|18s rRNA |23.5 ± 2.6 |23.0 ± 1.7 |0.2 |23.6 ± 3.9 |23.3 ± 3.6 |0.2 |

Abbreviations: 18srRNA-Ribosomal RNA 18s, LA-Left Atrial, RA-Right Atrial, AF-Atrial Fibrillation, NSR-Normal Sinus Rhythm

Table 5. Delta cT values from Left and Right Atrial Samples

|Gene |LA |RA |LA-RA Fold Difference |p value |

| |N = 44 |N = 44 | | |

|COL-I |5.2 ± 2.3 |4.7 ± 4.8 |0.75 |0.3 |

|COL-III |5.0 ± 3.2 |5.8 ± 5.4 |1.8 |0.2 |

|TGF-β |7.5 ± 2.6 |6.9 ± 5.1 |0.6 |0.2 |

|PDGF |9.5 ± 2.9 |8.9 ± 4.3 |0.7 |0.2 |

|ANG |7.9 ± 3.0 |7.0 ± 5.5 |0.5 |0.2 |

|TIMP-1 |6.1 ± 3.8 |9.3 ± 5.1 |9.1 |0.002 * |

|MMP-1 |10.4 ± 3.0 |9.3 ± 7.0 |0.5 |0.2 |

|MMP-9 |11.7 ± 3.1 |10.1 ± 6.6 |0.3 |0.08 |

Abbreviations: Collagen type I- Col-1; Collagen type III- COL-III; Transforming Growth Factor Beta-TGF-β; Platelet Derived Growth Factor-PDGF; Angiotensin II-ANG; Tissue Inhibitor of Metalloproteinase I-TIMP-1; Matrix Metalloproteinase 1-MMP-1; Matrix Metalloproteinase 9; MMP-9, LA-Left Atrial, RA-Right Atrial

• Denotes statistical significance using paired Student t test.

Table 6. Subject Characteristics of Healthy Controls and Patients Having Surgery

| |Healthy Controls (n = 10) |NSR (n = 36) |AF (n = 18) |P value |

|Age |41.4 ± 12.8 |64.5 ± 8.5 |73.3 ± 7.8 | ................
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