There are no approved drugs for the treatment of heart failure with preserved ejection fraction (HFpEF), which is characterized by left ventricular (LV) diastolic dysfunction. We demonstrate that ITF2357 (givinostat), a clinical-stage inhibitor of histone deacetylase (HDAC) catalytic activity, is efficacious in two distinct murine models of diastolic dysfunction with preserved EF. ITF2357 blocked LV diastolic dysfunction due to hypertension in Dahl salt-sensitive (DSS) rats and suppressed aging-induced diastolic dysfunction in normotensive mice. HDAC inhibitor–mediated efficacy was not due to lowering blood pressure or inhibiting cellular and molecular events commonly associated with diastolic dysfunction, including cardiac fibrosis, cardiac hypertrophy, or changes in cardiac titin and myosin isoform expression. Instead, ex vivo studies revealed impairment of cardiac myofibril relaxation as a previously unrecognized, myocyte-autonomous mechanism for diastolic dysfunction, which can be ameliorated by HDAC inhibition. Translating these findings to humans, cardiac myofibrils from patients with diastolic dysfunction and preserved EF also exhibited compromised relaxation. These data suggest that agents such as HDAC inhibitors, which potentiate cardiac myofibril relaxation, hold promise for the treatment of HFpEF in humans.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca 2+ -activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca 2+ -activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm 2 (resting tension), 146.8 ± 13.8 mN/mm 2 (maximal active tension, P 0 ), 5.4 ± 0.22 s −1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s −1 (linear relaxation rate), and 10.8 ± 1.3 s −1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa 50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa 50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca 2+ -sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and cells. The cell-stretcher, a novel device for the mechanical uniaxial stimulation of populations of cells, is described. The system is based on a variable stroke cam-lever-tappet mechanism which allows the delivery of cyclic stimuli with frequencies of up to 10 Hz and deformation between 1% and 20%. The kinematics is presented and a simulation of the dynamics of the system is shown, in order to compute the contact forces in the mechanism. The cells, following cultivation and preparation, are plated on an ad hoc polydimethylsiloxane membrane which is then loaded on the clamps of the cell-stretcher via force-adjustable magnetic couplings. In order to show the viability of the experimentation and biocompatibility of the cell-stretcher, a set of two in vitro tests were performed. Human epithelial carcinoma cell line A431 and Adult Mouse Ventricular Fibroblasts (AMVFs) from a dual reporter mouse were subject to 0.5 Hz, 24 h cyclic stretching at 15% strain, and to 48 h stimulation at 0.5 Hz and 15% strain, respectively. Visual analysis was performed on A431, showing definite morphological changes in the form of cellular extroflections in the direction of stimulation compared to an unstimulated control. A cytometric analysis was performed on the AMVF population. Results show a post-stimulation live-dead ratio deviance of less than 6% compared to control, which proves that the environment created by the cell-stretcher is suitable for in vitro experimentation.

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BackgroundCardiac hypertrophy can be considered a maladaptive response which in many cases results in heart failure and sudden death. Genetic predisposition to cardiac hypertrophy is ill-defined, but current research has given credence to a role of non-protein-coding RNAs in the development of cardiac hypertrophy.ResultsWe used microarrays, RNA-Seq and quantitative genetics with the spontaneously hypertensive, SHR (SHR/OlaIpcv) rat strain, the normotensive Brown Norway (BN-Lx/Cub) rat strain, and a recombinant inbred (RI) panel of rats (HXB/BXH) derived from these strains to examine the areas of the genome associated with cardiac hypertrophy. We identified circular (circ) RNAs coded within these areas. Of the 122 differentially expressed circRNAs in left ventricle of SHR and BN-Lx rats, three were transcribed from areas corresponding to the QTLs we identified for cardiac hypertrophy using the HXB/BXH rat panel. We then identified microRNAs which are “sponged” by the circ RNAs and the mRNAs which are destabilized by the microRNAs. 265 miRNAs could be identified as targets for the three circRNAs. We focused on the four miRNAs that were also differentially expressed between SHR and BN-Lx rats. One of the miRNAs (Mir-210-5p) was a target of the differentially expressed circ H2afy and circ H2afy was located within the QTL on Chr 17 (genome wide p-value = 0.011). Mir-210-5p has a binding site on the 3’ UTR of DR6, which is differentially expressed and in turn controls the expression level of NFκB. NFκB is an important component of the oxidative stress response leading to hypertrophy.ConclusionsOur work identified a novel circRNA that may be the mediator of a cascade of events that influence a cardiac hypertrophy phenotype. The circRNA and miRNA which we identified may become useful as markers of the risk for cardiac hypertrophy.