Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy

Abstract

Background: Recent work has demonstrated the importance of chromatin remodeling, especially histone acetylation, in the control of gene expression in the heart. In cell culture models of cardiac hypertrophy, pharmacological suppression of histone deacetylases (HDACs) can either blunt or amplify cell growth. Thus, HDAC inhibitors hold promise as potential therapeutic agents in hypertrophic heart disease.

Methods and results: In the present investigation, we studied 2 broad-spectrum HDAC inhibitors in a physiologically relevant banding model of hypertrophy, observing dose-responsive suppression of ventricular growth that was well tolerated in terms of both clinical outcome and cardiac performance measures. In both short-term (3-week) and long-term (9-week) trials, cardiomyocyte growth was blocked by HDAC inhibition, with no evidence of cell death or apoptosis. Fibrotic change was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was blocked. Preservation of systolic function in the setting of blunted hypertrophic growth was documented by echocardiography and by invasive pressure measurements. The hypertrophy-associated switch of adult and fetal isoforms of myosin heavy chain expression was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor-treated hearts.

Conclusions: Together, these data suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of load-induced heart disease.

Figure 1

TSA provokes histone acetylation in vitro and in vivo. A, Chemical structure of TSA. B, Neonatal cardiomyocytes in culture were exposed to 100 nmol/L TSA (48 hours) followed by immunoblot analysis for acetyl-histone H3 and acetyl-tubulin. C, Mice were exposed to TSA 1 mg/kg, and then LV lysates were subjected to immunoblot analysis for histone H3. TSA caused a significant increase in acetylation of histone H3. D, LV lysates from hearts treated as listed (3 weeks) were subjected to immunoblot analysis. Both TSA and TAB triggered histone H3 acetylation. E, Densitometric quantification of acetyl-H3 abundance, normalized to glyceraldehyde-3-phosphate dehydrogenase. * P<0.05 versus Veh.

Figure 2

TSA blunts pressure-overload hypertrophy. A, Representative 4-chamber histological sections of hearts treated as listed. B, Heart mass (HW) or LV mass normalized to body mass (BW) in hearts treated as listed. Sham+Veh (n=30, black bar); sham+TSA (n=28); TAB+Veh (n = 19, solid gray bar); TAB+TSA (0.5 mg/kg, n=5, lightly stippled bar); TAB+TSA (1 mg/kg, n = 18, stippled bar); TAB+TSA (2 mg/kg, n=5, heavily stippled bar). *P<0.05 versus sham; ** P<0.05 versus TAB+Veh. C, Representative fields of Mas-son’s trichrome–stained tissue sections reveal interstitial fibrosis typical of pressure-overload-induced hypertrophy (left) is nearly abolished by TSA (right). D, Fibroblasts were isolated from adult murine left ventricle and exposed to TGF-β (5 ng/mL) for 24 hours in the presence/absence of TSA. A representative immunoblot probed for type I collagen is shown, revealing dose-responsive suppression of collagen biosynthesis. Cntl indicates control.

Figure 3

TSA blunts cardiomyocyte growth and preserves natriuretic peptide expression. A, Two-dimensional cardiomyocyte cross-sectional area measured as described. Sham+Veh (n=470 cells/5 hearts); sham+TSA (n=460/4); TAB+Veh (n=560/4); TAB+TSA (n=500/4). B, Transcript abundance was quantified by Northern dot blot analysis (n=3 independent experiments), revealing persistently elevated level of ANF and BNP transcript in TAB+TSA ventricle.

Figure 4

Scriptaid (SA) blunts pressure-overload hypertrophy. Heart mass (HW) or LV mass normalized to body mass (BW). Sham+Veh (n=7, black bar); sham+SA (n=8); TAB+Veh (n=7, solid gray bar); TAB+SA (2.5 mg/kg, n=4, lightly stippled bar); TAB+SA (5 mg/kg, n=6, heavily stippled bar). Inset, SA chemical structure. * P<0.05 versus sham; ** P<0.05 versus TAB+Veh.

Figure 5

A, Kaplan-Meier analysis demonstrated similar survival in TAB animals treated with vehicle (open squares, n=42) or TSA (filled squares, n=50). Echocardiographic measures of systolic performance (B) and ventricular size (C) reveal preservation of cardiac function in TSA-treated animals. C, Black bars depict diastolic dimensions and gray bars depict systolic dimensions.

Figure 6

TSA abolished hypertrophy-associated decreases in LV performance. A, Representative pressure-volume loop recordings from mice treated as listed (3 weeks). ESPVRs were calculated using a computerized algorithm. B, Mean ESPVR data. Sham+Veh (n=4); sham+TSA (n=6); TAB+Veh (n=8); TAB+TSA (n=8). C, Mean data from maximal (white bars) and minimal (black bars) dP/dt measurements.

Figure 7

Blunted hypertrophy-associated switching of MHC isoforms in hearts exposed to TSA. Representative immunoblots from individual left ventricles treated as listed and probed for β-MHC (A), α-MHC (B), or α-tubulin (C).

Figure 8

Long-term treatment with TSA is well tolerated. A, HW/BW ratios in hearts treated for 9 weeks as listed. Sham+Veh, n=7; sham+TSA, n=7; TAB+Veh, n=11; TAB+TSA, n=13. B, Percent fractional shortening measured at the end of the 9-week trial reveals statistically significant attenuation of pressure overload-induced systolic dysfunction in TAB hearts treated with TSA or vehicle. C, Representative fields of Masson’s trichrome-stained tissue sections reveal marked accumulation of interstitial fibrosis (left) which is nearly abolished by TSA (right). D, Two-dimensional cardiomyocyte cross-sectional area measured as described. Sham+Veh (n=552 cells/3 hearts); sham+TSA (n=580/3); TAB+Veh (n=634/3); TAB+TSA (n=658/3).