**2.5 Statistics**

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

**Forward Reverse** ANP ATG GGC TCC TTC TCC ATC AC TCT TCG GTA CCG GAA GCT BNP ATG ATT CTG CTC CTG CTT TTC TCT GCA TCG TGG ATT GTT CTG MHC-α CAC CCT GGA GGA CCA GAT TA TGG ATC CTG ATG AAC TTC CC TGF-β<sup>1</sup> ATT CCT GGC GTT ACC TTG G CCT GTA TTC CGT CTC CTT GG Biglycan TGA TTG AGA ATG GGA GCC TGA G CCT TGG TGA TGT TGT TGG AGT G Decorin GGC AGT CTG GCT AAT GTT C CTT CGG AGA TGT TGT TGT TAT G Collagen-1 GCG AAC AAG GTG ACA GAG CCA GGA GAA CCA GCA GAG Collagen-3 TGG AGT CGG AGG AAT G GCC AGA TGG ACC AAT AG Elastin TGC TAC TGC TTG GTG GAG AAT G CGT GGC TGC TGC TGT CTG Fibronectin TGG AGC AAG AAG GAC AAC CGG ACA TCT GTG AAG GAG Laminin CGA GGA TGT CAG CGT TGT TCA CAG CCG TCT CCA GTC

SERCA2a CGA GTT GAA CCT TCC CAC AA AGG AGA TGA GGT AGC GGA TGA A Phospholamban TAT GTC TGC TGC TGA TAT GC ACT CTT AAA TCG TGA CCC TTC NCX CGC TAA TCA GCA TTT CAG AG GCC AGG TTC GTC TTC TTA AT Bax ACT AAA GTG CCC GAG CTG ATC CAC TGT CTG CCA TGT GGG G Bcl-2 ATC TTC TCC TTC CAG CCT GA TCA GTC ATC CAC AGA GCG AT ODC GAA GAT GAG TCA AAC GAG CA AGT AGA TGT TTG GCC TCT GG Arginase-2 TGA GGA GCA GCG TCT CCC GT GCT TCT CGG ATG GCG GCT GG eNOS AGC CCG GGA CTT CAT CAA TCA G GCC CCA AAC ACC AGC TCA CTC TC

Intermedin TGC CTC AGG GTG GTG GCT CAA CT GTG GGG GCT GCT GGG AT RAMP-1 AGC ATC CTC TGC CCT TTC ATT GAC CAC CAG GGC AGT CAT G RAMP-2 GCA GCC TAC CTT CTC CGA TCC TCC TCC ACA CCA CAA GCG TAA C RAMP-3 CAA CCT GTC GGA GTT CAT CGT TGT CTC CAT CTC CGT GCA GTT MMP9 CAA TCC TTG CAA TGT GGA TG AAA TCT TCT TGG ACT GCG GA MMP12 TGC AGC TGT CTT TGA TCC AC GCA TCA ATT TTT GGC CTG AT iNOS AAG AGA CGC ACA GGC AGA G CAG CAG GCA CAC GCA ATG Nrf-1 GGC ATC ACT GGC AGA GGC CG GCT GCT GCG GTT TCC CCA GA PGC-1α AGT GCT CAG CCG AGG ACA CGA TGC CCC TGC CAG TCA CAG GA SDF-1α CCA AGG TCG TCG CCG TGC TG GGC TCT GGC GAC ATG GCT CT VEGF TGC CCC TAA TGC GGT GTG CG GGC TCA CAG TGA ACG CTC CAG G GATA4 CTA TGG CCG CCA ACC ACG GG CGC GGA GTG GGC ACG TAG AC Mef-2c CAG TTG GGA GAC CGT ACC AC GTG AGT CCA ATG GGG GAG TG Nkx.2a CAC ACG CCC TCC TCA GTC AA GAG TAG CCG TCC GGC TTG AA vWF AAG ATG GCA AGA GAG TGG GC CCG TAG GCC TCA CTG GAA AG VE-cadherin CCA GAA TTT GCC CAG CCC TA GTC CTC GTT CTT CAG GGC AA B2M GCC GTC GTG CTT GCC ATT C CTG AGG TGG GTG GAA CTG AGA C HPRT CCA GCG TCG TGA TTA GTG AT CAA GTC TTT CAG TCC TGT CC

**10**

**Table 1.**

*List of primers used in this study.*

The results are expressed as means ± S.E.M or median with 25 and 75% quartiles as indicated in the legend to the figures. Statistical comparisons were performed by two-side T-Test or Mann-Whitney Test. Levene test was used to check the normal distribution of the samples. A p value of 0.05 was considered as statistical significant.

## **3. Results**

### **3.1 Weight of organs, left ventricular pressures, and biventricular functions analysis over time**

Body weight, LV weight, RV weight, lung wet weight, and kidney weight increased during the 4-months observation period in sham rats and those undergoing ischemia/reperfusion (**Table 2**). Mean increase in body weight at day 3 after the ischemic event was smaller in the surgery group (+2 g) versus the sham group (+13 g) indicating a small impact of the surgery on general behavior. Differences between both groups in ventricular weight occurred only for the RV at day 1 (**Table 1**). RV weights of rats in the experimental group normalized thereafter. No differences were obtained for lung and kidney weights (**Table 2**). Necrotic tissue was nearly exclusively seen in LV (**Figure 1**).

LV function was determined in vitro. Immediately after ischemia/reperfusion, a significant decline in cardiac function was observed but this was normalized thereafter (**Table 3**). Biventricular function was analyzed after 120 days via echocardiography. RV fractional area change (four-chamber view) and LV ejection fraction (longitudinal four-chamber view) were significantly lower in rats of the experimental group compared to shams (**Table 4**).

#### **3.2 Biventricular gene regulation over time**

In total, biventricular expression of 36 genes was analyzed by a real-time RT-PCR. These genes cover the following area of interest: cardiac hypertrophy (ANP, BNP, MHC-α), fibrosis (TGF-β1, biglycan, decorin, collagen-1, collagen-3, elastin, fibronectin, laminin, MMP9), intracellular calcium handling (SERCA2a, phospholamban, NCX), apoptosis (bax, bcl-2), arginine metabolism (ODC, arginase-2, eNOS), receptor coupling (intermedin, RAMP-1, -2, -3), inflammation (iNOS, MMP12), cardiac metabolism (Nrf-1, PGC-1α), stem cell mobilization (SDF-1α, CXCR4, VEGF), cardiac transcription factors (GATA-4, Mef-2c, Nkx.2a), and endothelial markers (von Willebrand factor (vWF), VE-cadherin). **Figure 2** shows how many of these 36 genes were either


#### *Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

*Data are means ± S.D. from n = 8–9 rats. Data are given for body weight (BW), left ventricular weight (LV), right ventricular weight (RV), lung weight, and kidney weight. Data for LV, RV, lung, and kidney were normalized to body weight.*

#### **Table 2.**

*Body weight and organ weight.*


#### **Table 3.**

*Left ventricular developed pressure (LVDP).*


*Data are means ± S.D. from n = 9–13 rats. LV EF = left ventricular ejection fraction (determined by four-chamber longitudinal view); RV FAC = right ventricular fractional area change (determined by four-chamber view).*

#### **Table 4.**

*Biventricular function determined by echocardiography.*

upregulated or downregulated in the two ventricles over the time. In general, there was an upregulation of several genes mainly at day 3 postinfarction, whereas a downregulation of genes dominated at days 7 and 120. At the first time point (day 1),

**13**

**Figure 2.**

*Right Heart Adaptation to Left Ventricular STEMI in Rats*

there were significant differences in the expression between both ventricles. In the LV, four genes were significantly downregulated: GATA4, VEGF, eNOS, and MHC-α. VEGF, eNOS, and MHC-α genes contain a GATA4 promoter region. As expected, GATA4 expression correlated significantly with that of VEGF, eNOS, and MHC-α (**Figure 3**). Only two genes were upregulated (MMP12, Mef2c) at that time point. As MMP12 is a neutrophil-specific elastase, its strong expression in LV may indicate leukocyte infiltration into the LV during initial tissue repair. In the RV, only one gene was significantly affected by myocardial ischemia/reperfusion at that time (NCX). **Figure 4** shows the relative expression of all genes including those that were either up- or downregulated but without reaching the level of significance. At day 1, there is more variability and, therefore, more transcriptional adaptation in LV compared to RV. In the LV, the strongest downregulation that was not yet significant (>0.05) was found for laminin (again correlated with GATA4; **Figure 3**). The strongest upregulation that was not yet significant was found for MMP9. However, lack of statistical significance for laminin and MMP9 indicates a high interindividual variability. In the RV, ANP and Nkx.2a were strongly upregu-

lated and collagen-3 was strongly downregulated but yet not significant.

*Time-dependent induction gene regulation in both ventricles. Note that the number of genes differentially* 

*regulated (cutoff p < 0.05) is increased in RV after 3 days.*

*DOI: http://dx.doi.org/10.5772/intechopen.84868*

#### *Right Heart Adaptation to Left Ventricular STEMI in Rats DOI: http://dx.doi.org/10.5772/intechopen.84868*

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

**BW LV/BW RV/BW Lung/BW Kidney/BW (g) (mg/g) (mg/g) (mg/g) (mg/g)**

Sham 208 ± 28 2.76 ± 0.37 0.48 ± 0.10 8.38 ± 2.06 5.87 ± 0.65 I/R 209 ± 16 2.87 ± 0.16 0.66 ± 0.07 8.90 ± 3.01 6.94 ± 2.11 P value 0.958 0.317 0.003 0.711 0.222

Sham 222 ± 17 2.64 ± 0.11 0.57 ± 0.06 8.14 ± 2.00 6.05 ± 0.39 I/R 211 ± 21 2.69 ± 0.22 0.64 ± 0.10 7.28 ± 0.77 5.77 ± 0.25 P value 0.292 0.550 0.135 0.674 0.133

Sham 222 ± 20 2.69 ± 0.15 0.51 ± 0.08 7.47 ± 0.95 5.80 ± 0.36 I/R 225 ± 22 2.93 ± 0.25 0.51 ± 0.07 7.81 ± 1.43 6.03 ± 0.61 P value 0.775 0.054 1.000 0.613 0.389

Sham 255 ± 16 2.69 ± 0.13 0.53 ± 0.06 8.84 ± 1.81 5.85 ± 0.34 I/R 256 ± 21 2.74 ± 0.52 0.55 ± 0.12 9.89 ± 1.08 6.32 ± 0.73 P value 0.908 0.736 0.666 0.211 0.210 *Data are means ± S.D. from n = 8–9 rats. Data are given for body weight (BW), left ventricular weight (LV), right ventricular weight (RV), lung weight, and kidney weight. Data for LV, RV, lung, and kidney were normalized to body* 

Day 1 157.5 ± 20.9 mmHg 122.9 ± 21.7 mmHg Δ = −34.6 mmHg 0.006 Day 3 155.0 ± 23.8 mmHg 132.0 ± 30.1 mmHg Δ = −23.0 mmHg 0.113 Day 7 188.2 ± 31.4 mmHg 162.1 ± 15.4 mmHg Δ = −26.1 mmHg 0.054 Day 120 144.4 ± 10.4 mmHg 137.9 ± 22.3 mmHg Δ = −6.5 mmHg 0.442

LV EF 65.0 ± 5.8 51.3 ± 8.1 Δ = −13.7% 0.000 RV FAC 55.8 ± 7.2 45.5 ± 7.2 Δ = −10.3% 0.004 *Data are means ± S.D. from n = 9–13 rats. LV EF = left ventricular ejection fraction (determined by four-chamber longitudinal view); RV FAC = right ventricular fractional area change (determined by four-chamber view).*

**Sham I/R Δ P value**

**Sham I/R Δ P value**

upregulated or downregulated in the two ventricles over the time. In general, there was an upregulation of several genes mainly at day 3 postinfarction, whereas a downregulation of genes dominated at days 7 and 120. At the first time point (day 1),

**12**

Day 1

Day 3

Day 7

Day 120

*weight.*

**Table 2.**

**Table 3.**

**Table 4.**

*Body weight and organ weight.*

*Data are means ± S.D. from n = 8–9 hearts.*

*Left ventricular developed pressure (LVDP).*

*Biventricular function determined by echocardiography.*

there were significant differences in the expression between both ventricles. In the LV, four genes were significantly downregulated: GATA4, VEGF, eNOS, and MHC-α. VEGF, eNOS, and MHC-α genes contain a GATA4 promoter region. As expected, GATA4 expression correlated significantly with that of VEGF, eNOS, and MHC-α (**Figure 3**). Only two genes were upregulated (MMP12, Mef2c) at that time point. As MMP12 is a neutrophil-specific elastase, its strong expression in LV may indicate leukocyte infiltration into the LV during initial tissue repair. In the RV, only one gene was significantly affected by myocardial ischemia/reperfusion at that time (NCX). **Figure 4** shows the relative expression of all genes including those that were either up- or downregulated but without reaching the level of significance. At day 1, there is more variability and, therefore, more transcriptional adaptation in LV compared to RV. In the LV, the strongest downregulation that was not yet significant (>0.05) was found for laminin (again correlated with GATA4; **Figure 3**). The strongest upregulation that was not yet significant was found for MMP9. However, lack of statistical significance for laminin and MMP9 indicates a high interindividual variability. In the RV, ANP and Nkx.2a were strongly upregulated and collagen-3 was strongly downregulated but yet not significant.

**Figure 2.**

*Time-dependent induction gene regulation in both ventricles. Note that the number of genes differentially regulated (cutoff p < 0.05) is increased in RV after 3 days.*

#### **Figure 3.**

*Correlation between GATA4 expression and that of GATA4-dependent regulated genes.*

At day 3, there were many genes significantly upregulated in both ventricles such as BNP, MHC-α, SERCA2a, phospholamban, NCX, and VEGF. ANP and PGC-1α were also significantly upregulated but in both ventricles. However, the individual variability in RV was strong for these genes so that the level of induction did not reach the level of significance. An exception from the coregulation of genes between both ventricles is the transcription factor Nkx.2a. Nkx2a was upregulated in the LV but beyond the level of detection in the RV. At day 3, only two genes were downregulated in the LV. These are RAMP-2, that was similarly downregulated in the RV and vWF that was slightly reduced in the RV. In addition to the aforementioned genes, six genes were specifically upregulated in the RV that were not induced in the LV. These were decorin, collagen-3, eNOS, RAMP-3, MMP12, and Nrf-1. The increased expression of most of these genes is indicated in the plot shown in **Figure 4**.

At day 7, the expression of most genes induced at early time points was normalized again. However, at that time point, many genes were significantly downregulated. In the LV, this holds for NCX, intermedin, PGC-1α, vWF, and VE-cadherin. Only VE-cadherin and intermedin were also downregulated in the RV; although due to the high individual variability of the RV, this does not reach the level of significance. Only biglycan was induced in the LV at this time point. The gene expression profile of the RV differed significantly from that of the LV at this time point. SERCA2a and NCX were still strongly induced. TGF-β1, bax, bcl-2, RAMP-1, RAMP-2, iNOS, Nrf-1, and CXCR4 were all significantly downregulated. The level of regulation strongly increased the level of regulation of these genes in the left ventricle. **Figure 4** summarizes the expression of all genes at that time point.

**15**

(**Figure 4**).

**Figure 4.**

*Above the horizontal bar differences are below p < 0.05.*

*Right Heart Adaptation to Left Ventricular STEMI in Rats*

At day 120, 6 out of 36 genes under investigation were downregulated in the LV. These were decorin, RAMP-1, MMP9, MMP12, GATA4, and vWF. Among them, only GATA4 and vWF were not similarly downregulated in the RV. Elastin, fibronectin, bax, and Nkx.2a were specifically downregulated in the RV. Except for Nkx.2a, similar changes (although not significant) were also seen in the LV. The expression of SERCA2a was induced in the RV at that time but not in the LV. Overall, a stronger transcription adaptation is seen in the RV at that time

*Profile of gene expression at days 1, 3, 7, and 120 in both ventricles. Upregulated genes are on the right side.* 

*DOI: http://dx.doi.org/10.5772/intechopen.84868*

*Right Heart Adaptation to Left Ventricular STEMI in Rats DOI: http://dx.doi.org/10.5772/intechopen.84868*

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

At day 3, there were many genes significantly upregulated in both ventricles such as BNP, MHC-α, SERCA2a, phospholamban, NCX, and VEGF. ANP and PGC-1α were also significantly upregulated but in both ventricles. However, the individual variability in RV was strong for these genes so that the level of induction did not reach the level of significance. An exception from the coregulation of genes between both ventricles is the transcription factor Nkx.2a. Nkx2a was upregulated in the LV but beyond the level of detection in the RV. At day 3, only two genes were downregulated in the LV. These are RAMP-2, that was similarly downregulated in the RV and vWF that was slightly reduced in the RV. In addition to the aforementioned genes, six genes were specifically upregulated in the RV that were not induced in the LV. These were decorin, collagen-3, eNOS, RAMP-3, MMP12, and Nrf-1. The increased expres-

At day 7, the expression of most genes induced at early time points was normalized again. However, at that time point, many genes were significantly downregulated. In the LV, this holds for NCX, intermedin, PGC-1α, vWF, and VE-cadherin. Only VE-cadherin and intermedin were also downregulated in the RV; although due to the high individual variability of the RV, this does not reach the level of significance. Only biglycan was induced in the LV at this time point. The gene expression profile of the RV differed significantly from that of the LV at this time point. SERCA2a and NCX were still strongly induced. TGF-β1, bax, bcl-2, RAMP-1, RAMP-2, iNOS, Nrf-1, and CXCR4 were all significantly downregulated. The level of regulation strongly increased the level of regulation of these genes in the left ventricle. **Figure 4** summarizes the expression of all genes at that time point.

sion of most of these genes is indicated in the plot shown in **Figure 4**.

*Correlation between GATA4 expression and that of GATA4-dependent regulated genes.*

**14**

**Figure 3.**

**Figure 4.**

*Profile of gene expression at days 1, 3, 7, and 120 in both ventricles. Upregulated genes are on the right side. Above the horizontal bar differences are below p < 0.05.*

At day 120, 6 out of 36 genes under investigation were downregulated in the LV. These were decorin, RAMP-1, MMP9, MMP12, GATA4, and vWF. Among them, only GATA4 and vWF were not similarly downregulated in the RV. Elastin, fibronectin, bax, and Nkx.2a were specifically downregulated in the RV. Except for Nkx.2a, similar changes (although not significant) were also seen in the LV. The expression of SERCA2a was induced in the RV at that time but not in the LV. Overall, a stronger transcription adaptation is seen in the RV at that time (**Figure 4**).
