HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling

Abstract

Background: Diastolic dysfunction (DD) is associated with the development of heart failure (HF) and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation (AF). Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. Here, we addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction (EF). Methods: Four weeks following uninephrectomy (UNX) and implantation with deoxycorticosterone acetate (DOCA) pellets, when DD was clearly evident, one cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and endpoint invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo . Cardiac fibrosis was evaluated by picrosirius red (PSR) staining and second harmonic generation (SHG) microscopy of left ventricular (LV) sections, RNA-sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy (AFM) determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. Results: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. Surprisingly, in contrast to prior models, myofibril relaxation was unimpaired in UNX/DOCA mice. Furthermore, cardiac fibrosis was not evident in any mouse cohorts based on PSR staining or SHG microscopy. However, mass spectrometry revealed induction in the expression of more than one hundred extracellular matrix (ECM) proteins in LVs of UNX/DOCA mice, which correlated with profound tissue stiffening based on AFM. Remarkably, ITF2357/Givinostat treatment blocked ECM expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the pro-fibrotic chromatin reader protein, BRD4, to key gene regulatory elements. Conclusions: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD, and establish blockade of ECM remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Additionally, our data reveal the existence of pathophysiologically relevant 'covert' or 'hidden' cardiac fibrosis that is below the limit of detection of histochemical stains such as PSR, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Therapeutic targets for cardiac fibrosis: from old school to next-gen

“…More recently, it was shown that pan-HDAC inhibition with the clinical-stage compound ITF2357/givinostat improved cardiac relaxation in murine models of hypertension- or aging-induced DD with preserved ejection fraction, and that SAHA was efficacious in a feline model of HFpEF due to slow, progressive ascending aortic banding ( 107 – 109 ). Surprisingly, in the murine models of DD, cardiac fibrosis was not observed by standard histological readouts, such as Picrosirius red staining ( 107 , 108 ), and improved diastolic function upon HDAC inhibition was attributed exclusively to augmented myofibril relaxation ( 107 ). However, further evaluation of hearts of mice with DD revealed “hidden fibrosis,” a process in which increased ECM deposition and remodeling were not detected by standard histological methods, but were uncovered by quantitative mass spectrometry and atomic force microscopy (AFM).…”

“…However, further evaluation of hearts of mice with DD revealed “hidden fibrosis,” a process in which increased ECM deposition and remodeling were not detected by standard histological methods, but were uncovered by quantitative mass spectrometry and atomic force microscopy (AFM). This covert type of cardiac fibrosis was profoundly inhibited by ITF2357/givinostat in a manner that correlated with improved diastolic function ( 108 ), implicating HDAC inhibition as a potential therapeutic strategy to combat HFpEF induced by pathological ECM remodeling and resulting ventricular stiffening.…”

“…Class I HDAC inhibition was also shown to stimulate expression of antifibrotic microRNA-133 (miR-133), leading to suppression of TAC-mediated cardiac fibrosis in mice ( 124 ). More recently, suppression of CF activation by HDAC inhibition was linked to mislocalization of the chromatin “reader” protein bromodomain-containing protein 4 (BRD4) (see below) ( 108 ).…”

“…HAT activity is required to create acetyl-marks at profibrotic enhancers that are subsequently bound by BRD4. Furthermore, there is evidence demonstrating that HDAC inhibition, which creates spurious acetyl-histone marks, results in mislocalization of BRD4 in the CF genome ( 108 ) and prevents HATs from properly acetylating certain gene regulatory elements in the heart ( 141 ), which may also involve altering genomic targeting of bromodomain-containing HATs.…”

Genetic and environmental determinants of diastolic heart function

“…Pre-clinical models of diastolic dysfunction are associated with alterations in left ventricular stiffness on atomic force microscopy that occur at the level of the cardiomyocyte sarcomere as well as due to extracellular matrix protein expansion 9 . Such tissue level changes can be assessed at macroscopic scale in human populations through analysis of diastolic mechanics.…”