Functional significance of the discordance between transcriptional profile and left ventricular structure/function during reverse remodeling

Abstract

To elucidate the mechanisms for reverse LV remodeling, we generated a conditional (doxycycline [dox] off) transgenic mouse tetracycline transactivating factor-TRAF2 (tTA-TRAF2) that develops a dilated heart failure (HF) phenotype upon expression of a proinflammatory transgene, TNF receptor-associated factor 2 (TRAF2), and complete normalization of LV structure and function when the transgene is suppressed. tTA-TRAF2 mice developed a significant increase in LV dimension with decreased contractile function, which was completely normalized in the tTA-TRAF2 mice fed dox for 4 weeks (tTA-TRAF2^dox4W). Normalization of LV structure and function was accompanied by partial normalization (~60%) of gene expression associated with incident HF. Similar findings were observed in patients with dilated cardiomyopathy who underwent reverse LV remodeling following mechanical circulatory support. Persistence of the HF gene program was associated with an exaggerated hypertrophic response and increased mortality in tTA-TRAF2^dox4W mice following transaortic constriction (TAC). These effects were no longer observed following TAC in tTA-TRAF2^dox8W, wherein there was a more complete (88%) reversal of the incident HF genes. These results demonstrate that reverse LV remodeling is associated with improvements in cardiac myocyte biology; however, the persistence of the abnormal HF gene program may be maladaptive following perturbations in hemodynamic loading conditions.

Figure 1. Characterization of tTA-TRAF2 mice.

(A) tTA-TRAF2 mice and LM controls were followed up to 8 weeks of age and were then randomized to receive doxycycline in the drinking water or no doxycycline for 4 weeks. (B) Photographs of whole hearts and sagittal sections of LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W mouse hearts at 12 weeks of age. (C) Heart weight–to–body weight ratio (HW/BW). (D) Left ventricular end-diastolic diameter (LVEDD).(E) LV fractional shortening percent (FS%). (F) LV wall thickness (LVWTh). (G) r/h ratio of LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W at 12 weeks of age (n = 6 mice/group). (H) Representative transmission electron micrographs from 12-week LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W mice at ×10,000 and ×20,000 magnification. Protein aggregates are enclosed by the circle. Statistical analysis was performed using 2-way ANOVA with Tukey post-hoc analysis. (*P ≤ 0.05 compared with LM control.) LM, littermate; LM^dox4W, LM mouse fed doxycycline for 4 weeks; r/h ratio, radius/wall thickness.

Figure 2. Myocyte and LV chamber contractile function of tTA-TRAF2 mice.

(A) Representative tracings of sarcomere shortening in isolated cardiac myocytes and (B) group data of sarcomere shortening from LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W mouse hearts at 12 weeks of age (5–6/cells/heart from 3 hearts/group). (C) Left ventricular developed pressure (LVDP). (D) Peak +dP/dt and (E) peak –dP/dt in 12-week LM (n = 10), tTA-TRAF2 (n = 8), LM^dox4W (n = 11), and tTA-TRAF2^dox4W (n = 5) mouse hearts. Statistical analysis performed using 2-way ANOVA with Tukey post-hoc analysis. (*P < 0.05 compared with LM controls.)

Figure 3. Myocardial fibrillar collagen content in tTA-TRAF2 mice.

(A) Representative picrosirius red staining (×400) for myocardial fibrillar collagen content and (B) group data for % collagen volume for LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W mouse hearts (9–10 fields analyzed for n = 3 mice/group). Statistical analysis performed using 2-way ANOVA with Tukey post-hoc analysis. (*P < 0.05 vs. LM control.)

Figure 4. Transcriptional profiling in 12-week LM, tTA-TRAF2, LM^dox4W, and tTA-TRAF2^dox4W mouse hearts.

(A) Venn diagram of normalized heart failure (HF) genes, persistently dysregulated HF genes, and new reverse remodeled genes (see text for details). (B) Unsupervised hierarchical clustering of changes in gene expression, and (C) principal component analysis (PCA) of changes in gene expression. (D) Illustration of persistently dysregulated and reversed HF genes in relation to their anatomic location within the cardiac myocyte. (E) Illustration of persistently dysregulated and reversed HF genes in gene modules relating to cardiac myocyte function (see text for details). The number of genes within each module is given in parenthesis (n = 4 hearts per group). Red indicates increased gene expression; blue indicates decreased gene expression relative to LM; Rev, reversed HF genes; PD, persistently dysregulated HF genes. Analysis performed with Partek Genomics Suite. LM, littermate; LM^dox4W, LM mouse fed doxycycline for 4 weeks; r/h ratio, radius/wall thickness, PC, principal component; PD, persistently dysregulated; Rev, reversed.

Figure 5. Effect of transaortic constriction (TAC) on LV structure and mortality in LM^dox4W and tTA-TRAF2^dox4W mice.

(A) LV mass index (LVMI), (B) LV wall thickness (LVWTh), (C) LV end-diastolic diameter (LVEDD), and (D) LV r/h ratio at 2 and 4 weeks after TAC (baseline n = 7, 2 weeks after TAC n = 8–9, 4 weeks after TAC n = 5). (E) Kaplan-Meier analysis of survival in LM^dox and tTA-TRAF2^dox mice following TAC (n = 12 LM^dox4W hearts and n = 14 tTA-TRAF2^dox4W hearts). Statistical analysis performed using a repeated measures analysis using mixed models methodology (See Methods section). (*P < 0.05)

Figure 6. Transcriptional profiling in 16-week LM, tTA-TRAF2, LM^dox8W, and tTA-TRAF2^dox8W mouse hearts.

(A) tTA-TRAF2 mice and LM controls were followed up to 8 weeks of age and were then randomized to receive doxycycline in the drinking water or no doxycycline for 8 weeks. (B) Venn diagram of normalized heart failure (HF) genes, persistently dysregulated HF genes, and new reverse remodeled genes (see text for details). (C) Unsupervised hierarchical clustering of changes in gene expression and (D) principal component analysis (PCA) of changes in gene expression. (E) Illustration of persistently dysregulated and reversed HF genes in relation to their anatomic location within the cardiac myocyte. (F) Illustration of persistently dysregulated and reversed HF genes in gene modules relating to cardiac myocyte function (see text for details). The number of genes within each module is given in parenthesis (n = 4 hearts per group). Red indicates increased gene expression; blue indicates decreased gene expression relative to LM; Rev, reversed HF genes; PD, persistently dysregulated HF genes. Analysis was performed using Partek Genomic Suite. PC, principal component; PD, persistently dysregulated; Rev, reversed.

Figure 7. Analysis of reversed and persistently dysregulated genes in 16-week LM, tTA-TRAF2, LM^dox8W, and tTA-TRAF2^dox8W mouse hearts.

(A) Number of persistently dysregulated and reversed heart failure (HF) genes after 4 and 8 weeks of doxycycline. (B) Percentage of reversed HF genes in functional cardiac myocyte gene modules after 4 and 8 weeks of doxycycline. (C) Percentage of persistently dysregulated HF genes in functional cardiac myocyte gene modules after 4 and 8 weeks of doxycycline (n = 4 hearts per group). Analysis was performed using Partek Genomic Suite.

Figure 8. Effect of transaortic constriction (TAC) on LV structure and mortality in LM^dox8W and tTA-TRAF2^dox8W mice.

(A) LV mass index (LVMI), (B) LV wall thickness (LVWTh), (C) LV end diastolic diameter (LVEDD), and (D) LV r/h ratio at 2 and 4 weeks after TAC (n = 7 hearts per group). (E) Kaplan Meier analysis of survival in LM^dox and tTA-TRAF2^dox mice following TAC (n = 7 LM^dox8W hearts and n = 7 tTA-TRAF2^dox8W hearts). Statistical analysis performed using a repeated measures analysis using mixed models methodology (see Methods section). (*P <0.05).

Figure 9. Transcriptional profiling in nonfailing (NF) human hearts (n = 8) and paired failing human dilated cardiomyopathy (DCM) hearts (n = 8 pairs) before and after LVAD.

(A) Venn diagram of normalized heart failure (HF) genes, persistently dysregulated HF genes, and new reverse remodeled genes (see text for details). (B) Unsupervised hierarchical clustering of changes in gene expression, and (C) principal component analysis (PCA) of changes in gene expression.(D) Number of persistently dysregulated and reversed HF cardiac myocyte genes. (E) Illustration of persistently dysregulated and reversed HF genes in gene modules relating to cardiac myocyte function (see text for details). The number of genes within each module is given in parenthesis. Red indicates increased gene expression; blue indicates decreased gene expression relative to LM; Rev, reversed HF genes; PD, persistently dysregulated HF genes. Analysis performed using Partek Genomics Suite. (F) Percentage of persistently dysregulated and reversed HF genes in functional cardiac myocyte gene modules after LVAD support. PC, principal component; PD, persistently dysregulated; Rev, reversed.