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. Author manuscript; available in PMC: 2026 Jun 23.
Published in final edited form as: Sci Transl Med. 2024 Jul 17;16(756):eadm8842. doi: 10.1126/scitranslmed.adm8842

Vasohibin inhibition improves myocardial relaxation in a rat model of heart failure with preserved ejection fraction

Deborah M Eaton 1,#, Benjamin W Lee 1,#, Matthew A Caporizzo 2,3,#, Amit Iyengar 4, Christina Y Chen 2, Keita Uchida 2, Guillaume Marcellin 5, Yoann Lannay 5, Alexia Vite 1, Kenneth C Bedi Jr 1, Claire F Brady 1, Julia N Smolyak 1, Danika Meldrum 4, Jessica Dominic 4, Noah Weingarten 4, Mrinal Patel 4, Andrew Belec 4, Khaled Hached 5, Pavan Atluri 4, Siem Van Der Laan 5, Benjamin L Prosser 2,*, Kenneth B Margulies 1,*
PMCID: PMC13285409  NIHMSID: NIHMS2107089  PMID: 39018366

Abstract

Heart Failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with increased myocardial stiffness and cardiac filling abnormalities. Prior studies implicated increased α-tubulin detyrosination, which is catalyzed by the vasohibin enzymes, as a contributor to increased stabilization of the cardiomyocyte microtubule network (MTN) and stiffness in failing human hearts. We explored whether increased MTN detyrosination contributed to impaired diastolic function in the ZSF1 obese rat model of HFpEF and designed a small molecule vasohibin inhibitor to ablate MTN detyrosination in vivo. Compared to ZSF1 lean and Wistar Kyoto rats, obese rats exhibited increased tubulin detyrosination concomitant with diastolic dysfunction, left atrial enlargement, and cardiac hypertrophy with a preserved left ventricle ejection fraction, consistent with a HFpEF phenotype. Ex vivo myocardial phenotyping assessed cardiomyocyte mechanics and contractility. Vasohibin inhibitor treatment of isolated cardiomyocytes from obese rats resulted in reduced stiffness and faster relaxation. Acute in vivo treatment with vasohibin inhibitor improved diastolic relaxation in ZSF1 obese rats compared to ZSF1 lean and Wistar Kyoto rats. Vasohibin inhibition also improved relaxation in isolated human cardiomyocytes from both failing and non-failing hearts. Our data suggest the therapeutic potential for vasohibin inhibition to reduce myocardial stiffness and improve relaxation in HFpEF.

Editor’s summary:

Relaxing the heart.

Heart failure with preserved ejection fraction (HFpEF) is a growing clinical concern with limited pharmacological options for treatment. HFpEF is characterized by reduced ability of the myocardium to relax properly during diastole. Eaton, Lee, and Caporizzo et al. developed and tested two vasohibin inhibitors that decreased α-tubulin detyrosination to improve cardiomyocyte relaxation. In a ZSF1-obese rat model of HFpEF, isolated cardiomyocytes had increased relaxation and decreased stiffness after vasohibin inhibition compared to cardiomyocytes from lean rats. In vivo vasohibin inhibition improved diastolic relaxation in these rat hearts. In cells from human hearts with or without heart failure, vasohibin inhibition resulted in improved relaxation in vitro. These results suggest that inhibiting detyrosination of microtubule networks in cardiomyocytes could be a target for treating HFpEF. ---Brandon Berry

One Sentence Summary:

Blocking α-tubulin detyrosination with a small-molecule inhibitor improves myocardial relaxation in the ZSF1-obese rat model of HFpEF.

INTRODUCTION

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome with a paucity of effective therapies compared to HF with reduced ejection fraction (HFrEF)(1), representing a major unmet clinical need. Patients with HFpEF are characterized as having left ventricular diastolic dysfunction(1),(2), with impaired relaxation and/or increased stiffness contributing to elevated cardiac filling pressures at rest and/or during exercise. Potential origins of myocardial diastolic dysfunction in HFpEF include extracellular elements, such as fibrosis (3) or infiltrating proteins as in amyloidosis (4), but also include pathologic adaptations within the cardiomyocyte such as titin isoform switching (5) or shifts in other cytoskeletal constituents.

The increased abundance and stabilization of the cardiomyocyte microtubule network (MTN) contributes to increased cardiomyocyte viscoelasticity and stiffness that impedes both contraction and relaxation in failing human hearts requiring transplantation (6, 7). These studies and others have demonstrated that MTN stabilization is at least in part mediated by detyrosination, which is the post-translational removal of the C-terminal tyrosine of α-tubulin (8–12). Specifically, detyrosination increases the association of microtubules with desmin at the Z-disc, leading to increased stiffness and viscoelasticity of the myocyte, and impedes both cardiomyocyte shortening and relengthening (6). The balance between detyrosinated and tyrosinated α-tubulin is regulated by enzymes with reciprocal actions: tubulin tyrosine ligase (TTL) and carboxypeptidases identified as vasohibins (VASH1 and VASH2) (13). The identification of VASHs as tubulin detyrosinases was made possible by employing a high affinity purification step using custom-designed inhibitors compatible with click chemistry(13). The most efficient among said inhibitors, referred to as EpoY(13), is composed of an active group called transepoxysuccinate (TES) ethyl ester linked to a tyrosine residue. However, this compound shows a high IC50 of about 15 μM in cells. A small and highly electrophilic epoxide molecule such as EpoY might interact with free thiols present on other proteins and in cysteine protease active sites at high concentrations. Additionally, it could react with nucleophilic lysine residues in tyrosine kinase active sites, resulting in a loss of specificity. Using medicinal chemistry, libraries of modified derivatives were generated and we measured their effect in an in vitro system and used bioinformatics to model and understand the structural properties responsible for their variations in biological activities. Ultimately, we have developed a new class of potent and specific small molecule VASH inhibitors (VASHi), which we characterize and test using translationally relevant ex vivo and in vivo models.

The ZSF1 obese rat, has two separate leptin receptor mutations as a result of crossing the lean female Zucker diabetic fatty rats and male spontaneously hypertensive heart failure rats. This model recapitulates several HFpEF risk factors, including obesity, diabetes, hypertension, and skeletal muscle dysfunction (14–20). ZSF1 lean rats are heterozygous for the leptin receptor mutations. In this study, we test the hypothesis that VASH-dependent MTN detyrosination contributes to impaired diastolic function in the ZSF1 obese rat model of HFpEF and can be targeted using a new small molecule VASH inhibitor.

RESULTS

ZSF1 Obese Rats Develop Features of HFpEF and Elevated Detyrosination

Figure 1A-B presents an initial survey of the temporal progression of diastolic dysfunction and myocardial tubulin detyrosination in WKY and ZSF1 obese rats. ZSF1 obese rats already exhibited elevated catheter-derived left ventricular end-diastolic pressure (LVEDP) and prolonged myocardial relaxation (Tau) at 10 weeks of age relative to WKY rats. Diastolic dysfunction appeared to worsen from 10–20 weeks of age, and remained evident but with no further progression from 20–30 weeks of age (Fig. 1A and B). Protein abundance of myocardial detyrosinated tubulin mirrored this time course (Fig. 1C and D), whereastotal tubulin (α-tubulin) abundance progressively increased in obese rats from approximately 10–25 weeks of age (Fig. 1E and F). These data support a close temporal link between myocardial detyrosination and diastolic dysfunction in ZSF1 obese rats. Additional hemodynamic parameters are reported in table S1. The following experiments in this study were performed on rats and isolated cardiomyocytes between 20–30 weeks of age.

Figure 1. ZSF1 Obese Rats Develop Features of HFpEF at 20 Weeks of Age with Increased Detyrosinated-Tubulin.

Figure 1.

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(A) Tau, left ventricular diastolic time constant, and (B) left ventricular end-diastolic pressure were assessed from the age of 10 weeks to 30 weeks in WKY and ZSF1 obese rats (N=6–12 rats per group). (C) Western blot images for protein abundance of detyrosinated tubulin and α-tubulin in left ventricle (LV) tissue from wky and obese rats aged 10 to 30 weeks, with age notated above blot. (D) Quantification of detyrosinated-tubulin, normalized to GAPDH, (E) α-tubulin, normalized to GAPDH, and (F) detyrosinated-tubulin, normalized to α-tubulin (N=6–12 rats per group). (G) Body weight, (H) mean arterial pressure, (I) heart weight to tibia length ratio, and (J) LV mass are shown for WKY, ZSF1 lean, and ZSF1 obese rats at 20 weeks of age (N=6–12 rats per group). (K) Tau, (L) mitral valve (MV) deceleration time, (M) left ventricle end-diastolic pressure (LVEDP), (N) left atrial (LA) area, (O) LV ejection fraction, and (P) tricuspid annular plane systolic excursion (TAPSE) are shown for WKY, ZSF1 lean, and ZSF1 obese rats at 20 weeks of age (N=6–12 rats per group). (Q) Western blot images from isolated cardiomyocytes show abundance of detyrosinated tubulin, α-tubulin, and GAPDH from rats grouped by younger (20–24 weeks) and older (27–30 weeks) age. (R) Quantification of the ratio of detyrosinated tubulin to GAPDH and (S) the ratio of α-tubulin to GAPDH (N=3–6 rats per phenotype). (T) Transcriptional changes in ZSF1 obese rats compared to ZSF1 lean rats (20 and 30 weeks of age) using NanoString with volcano plots of differentially expressed extracellular and tubulin associated transcripts are shown (N=3–6 rats per phenotype). Invasive hemodynamic derived parameters: A, B, H, K, M, O; Echocardiography-derived parameters: J, L, N, P. For G-S, significance was determined using one-way ANOVA. For T, significance was determined using an unadjusted paired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data shown are mean ± SEM.

Figure 1C-S presents more detailed characterization of the three rat strains (ZSF1 obese, ZSF1 lean, and WKY) at 20 weeks of age. As expected, obese ZSF1 rats had significantly higher body weights than lean and WKY rats (ob vs. ln, ob vs. wky: P<0.0001) (Fig. 1G). Obese rats also developed hypertension and significant cardiac hypertrophy compared to lean ZSF1 and WKY rats (ob vs. ln, ob vs. wky: P<0.0001) (Fig. 1H to J). Early diastolic function was impaired in ZSF1 obese rats compared to ZSF1 lean and WKY rats, based on significant prolongation of Tau (Fig. 1K) (ob vs. ln: P=0.001; ob vs. wky: P=<0.0001). Doppler-derived mitral valve (MV) deceleration time was not different between groups (Fig. 1L). LVEDP was increased in the ZSF1 obese rats compared to the ZSF1 lean and WKY rats, but also in the lean compared to the WKY (ob vs. ln: P=0.02; ob vs. wky: P<0.0001; ln vs. wky: P=0.04) (Fig. 1M). Both lean and obese ZSF1 rats exhibited increased left atrial area compared to WKY rats (Fig. 1N). All groups had preserved LV systolic function, measured using ejection fraction (Fig. 1O). The tricuspid annular plane systolic excursion (TAPSE), an indicator of global RV function, was preserved across all groups (Fig. 1P). Other echo and invasive hemodynamic parameters are presented in table S2.

Recognizing that patients with HFpEF have exaggerated increments in LV filling pressures with tachycardia, we explored responses to physiological stress. Transesophageal atrial pacing performed during the invasive hemodynamic study allowed us to directly measure LVEDP as the heart rate (HR) was increased. Obese rats had an initial decrease in LVEDP from an unpaced baseline to match the lean animals when pacing was started with a heart rate of 360 beats per minute (BPM) (fig. S1). Tachycardic pacing provoked a rate-dependent increase in LVEDP in obese animals. Lean and WKY rats had no change in LVEDP throughout the pacing protocol.

ZSF1 Obese Rat Cardiomyocyteshave Increased Detyrosinated Tubulin

We next examined MTN density in isolated cardiomyocytes from WKY, lean, and obese animals at 20–24 week and 27–30 week time points and confirmed a significant increase in total α-tubulin (P=0.01) and detyrosinated tubulin (P=0.002) in myocytes from obese rats compared to WKY (Fig. 1Q to S). To determine if extracellular matrix (ECM) or MTN-related transcripts were altered, we performed focused transcriptional profiling with a custom Nanostring gene expression panel. Among ECM genes, Vcan, Fn1 and Col1a1 were significantly increased in isolated cardiomyocytes of obese vs. lean ZSF1 rats (fig. S2). Among microtubule network genes and regulators, Tubb2a (P=0.0096), Vash1 (P=0.021), Hdac6 (P=0.0052) and Map1a (P=0.006) were significantly increased in obese vs. lean ZSF1 rats, with Vash1 exhibiting an approximately 2.2 fold increase in transcript expression (Fig. 1T, fig. S2).

Development of New Small Molecule VASH Inhibitors

We sought to generate potent and selective new chemical entities to inhibit VASH. Epo-Y had been previously characterized as a prototype VASH inhibitor. Specific peptide-based VASH Compounds (SVC-01 & SVC-02) are Epo-Y derivatives that were designed through multiple steps of chemical improvement to achieve potent inhibition of VASH as described (Patents WO2023025861 and WO2018EP69496) (13, 21). Chronologically, SVC-01 was developed first and after further refinement, SVC-02 was obtained.

Each compound (Fig. 2A) was tested on human CHL-1 cells for its capacity to reduce VASH-dependent tubulin detyrosination (22). Following 2 hours of treatment, the cells were fixed and processed for quantitative immunofluorescence staining of dTyr-Tubulin. Whereas Epo-Y yields a 50% inhibition of detyrosination at approximately 21 μM, the specific VASH compounds (SVC) SVC-01 and SVC-02 are new chemical entities, which presented with a calculated half-maximal inhibitory concentration (IC50) of 828 nM and 11 nM, respectively, up to 1974-fold more potent (Fig. 2B) than the previously published inhibitor (13).

Figure 2. Development of Small Molecule VASH Inhibitors.

Figure 2.

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(A) Chemical structures of Epo-Y and Specific VASH Compounds (SCVs) are shown. (B) The VASH activity in the human CHL-1 cell-line is expressed as a percentage relative to the vehicle control group. A dose-response experiment utilizing Epo-Y, SVC-01 and SVC-02 was conducted to determine their respective IC50 values. (C) VASH activity is determined in myocardial tissue treated ex-vivo with vehicle or SVC-02. The normalized VASH activity is expressed as a percentage relative to the vehicle control. (D) Western blot images for protein abundance (detyrosinated-tubulin, α-tubulin, and GAPDH) in myocardium dissected from Sprague Dawley rats 8 hours after IV injection of 6.6 mg/kg SVC-02. (E) Quantification of detyrosinated-tubulin/α-tubulin ratio and (F) detyrosinated-tubulin/GAPDH ratio (N = 4 Sprague Dawley rats treated with vehicle; N = 3 Sprague Dawley rats treated with SVC-02). For C, E, and F, statistical significance was determined using an unpaired t-test. ** P < 0.01, *** < 0.001. All data shown are mean ± SEM.

Recognizing that cathepsin B is a cysteine protease with features that may predict potential cross reactivity for VASH inhibitors (VASHi), SVC-01 was tested using a commercial cathepsin B screening kit (fig. S3A). Although the known cathepsin B inhibitor FF-FMK blocked the enzyme in a dose-dependent manner, SVC-01 did not inhibit cathepsin B, supporting specificity toward VASH. mRNA expression of detyrosinating and tyrosinating enzymes and accessory proteins in AC16 cells was unchanged after 8 hours of treatment, consistent with the inhibitor targeting enzyme activity, not expression (fig. S3B). Next a recombinant tubulin detyrosination assay with human VASH1 and VASH2 was performed with increasing concentrations of SVC-01. The activity of VASH1 and VASH2 were completely eliiminated with SVC-01 at a concentration of 10μM (fig. S3C) and no changes in protein abundance of VASH1 were observed at 4 and 8 hours of treatment (fig. S3D to F). We also evaluated the impact of VASH inhibition on another post-translational modification, acetylation, and found no effect compared to vehicle treatment (fig. S3G and H).

We then assessed the toxicity of SVC-01 in head-to-head comparisons to FDA-approved microtubule targeting agents (Parthenolide and Taxol). The CellTiter-Glo Luminescent Cell Viability Assay is a method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.. In comparison to vehicle treated cells, at day 2 and 3 we observed a reduction of at least 80% of the number of viable cells in presence of 10μM Taxol or Parthenolide, whereas SVC-01 treatment did not alter cell viability even at a concentration of 100μM for 3 days (fig. S4A). We also assessed mitochondrial activity using the Agilent Seahorse XF Cell Mito Stress Test Kit. Treatment of human CHL-1 cells with SVC-01 did not impact key parameters of mitochondrial function (fig. S4B).

We next tested efficacy of the most potent inhibitor to reduce tubulin detyrosination on freshly dissected hearts from Swiss mice. Overnight incubation with SVC-02 resulted in reduction (over 50% on average) in tubulin detyrosination in myocardial tissue (Fig. 2C). When SVC-02 was tested in vivo with Sprague Dawley rats treated by intravenous tail vein injection (6.6mg/kg), there was a significant reduction in the protein abundance of detyrosinated-tubulin to α-tubulin (P=0.0006) and GAPDH (P=0.0069) by over 50% compared to vehicle treatment (Fig. 2D to F). This timing scheme was based on a pilot study performed using Swiss Webster mice (fig. S3I and J).

VASH Inhibitor Reduces Detyrosinated Tubulin in Healthy Cardiomyocytes

For clarity, we will refer to SVC-01 and SVC-02 as VASH inhibitor (VASHi). We next tested whether VASHi could reduce tubulin detyrosination in cardiomyocytes isolated from healthy Sprague Dawley rats. Ex vivo treatment with VASHi (1.0–10.0μM) demonstrated a dose-dependent reduction in detyrosinated tubulin without affecting total α-tubulin abundance (Fig. 3A and B). We then evaluated the effect of VASHi compared to vehicle treatment on isolated, healthy cardiomyocyte contractility. In these healthy cardiomyocytes, we observed small, dose-dependent acceleration of contraction time and velocity (Figure 3C, D, E), as well as faster relaxation time and velocity (Fig. 3C, D, and F) between 0.1 and 5.0 μM, with no overt difference between 5 μM and 10 μM, suggesting saturation of the effect. There were no differences in resting sarcomere length nor fractional shortening with any dose of VASHi (Fig. 3G and H). Given these findings, 5 μM VASHi was used for the subsequent experiments in isolated cardiomyocytes.

Figure 3. VASH Inhibition Decreases Abundance of Detyrosinated Tubulin and Improves Contractility in Healthy Isolated Cardiomyocytes.

Figure 3.

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(A) A representative Western blot showing detyrosinated tubulin, α-tubulin, and GAPDH abundance of Sprague Dawley rat isolated cardiomyocytes treated ex vivo with 1 – 10 μM SVC-01. (B) Quantification of of α-tubulin (black) and detyrosinated tubulin (magenta) protein abundance in response to increasing concentrations of VASH inhibitor.(C) Average fractional shortening and (D) average velocities from cardiomyocytes treated with DMSO or increasing concentrations of VASHi (0.1 – 10 μM). (E) Contraction time, (F) relaxation time, (G) resting sarcomere length (SL) and (H) fractional shortening are shown (N = 3 Sprague Dawley rats, n = 50–60 myocytes per group). Statistical significance was determined using one-way ANOVA. * P < 0.05, ** P <0.01, **** P < 0.0001. All data shown are mean ± SEM.

VASH Inhibitor Improves Cardiomyocyte Relaxation in Obese ZSF1 Rats

We next evaluated the effect of VASHi on contractility in cardiomyocytes isolated from WKY, lean ZSF1, and obese ZSF1 rats at 30 weeks of age. Average fractional shortening and velocity tracings are shown in Figure 4A, B. Fractional shortening was similar in all three groups (Fig. 4A and C). Vehicle treated cardiomyocytes isolated from ZSF1-obese rats had both prolonged contraction time and time to 50% relaxation compared to lean and WKY, and the slowed kinetics were improved with VASHi treatment (Fig. 4B, D, and E). This improvement was equivalent to that observed with adenovirus-mediated overexpression of tubulin tyrosine ligase (TTL), an established method of reducing microtubule detyrosination in cardiomyocytes by overexpression of the enzyme that catalyzes the reverse reaction. Neither VASHi nor TTL completely restored cardiomyocyte relaxation and contraction times to the amounts observed in WKY and ZSF1 Lean cardiomyocytes. Super resolution images of vehicle and VASHi treated obese rat cardiomyocytes highlighted the near complete conversion of detyrosinated to tyrosinated microtubules at this time point, without overt changes in total microtubule network density (Fig. 4F). We also evaluated electrically evoked cytosolic Ca2+ transients in myocytes from ZSF1 lean and obese animals with and without VASHi. Though Ca2+ transients were slightly larger and prolonged in obese cardiomyocytes, VASHi had no effect on Ca2+ handling when compared to vehicle treatment (fig. S5), consistent with previous reports that used orthogonal approaches to acutely reduce detyrosination(23–25). This treatment-independent prolongation of the Ca2+ transient may contribute to the incomplete normalization of cardiomyocyte relaxation in VASHi treated ZSF1-obese cardiomyocytes, as may disease-associated abnormalities of titin-based stiffness.

Fig 4. Ex vivo VASH Inhibition Improves ZSF1 Obese Rat Cardiomyocyte Contractility.

Fig 4.

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For panels A and B, ZSF1 lean cardiomyocytes treated with vehicle are shown in gray or VASHi treated shown in light blue; ZSF1 obese cardiomyocytes treated with vehicle are shown in black or VASHi treated shown in dark blue. (A) Average fractional shortening and (B) velocity tracings from cardiomyocytes isolated from ZSF1 lean or ZSF1 obese rats and treated with vehicle or VASHi are shown. For panels C-E, vehicle treatment is shown in black, TTL treatment in pink, and VASHi in blue. (C) Fractional shortening, (D) contraction time, and (E) relaxation time to 50% are shown (N = 3 lean, N = 3 obese rats, n = ~90 myocytes per group; N = 2 WKY rats, n = 60 myocytes). (F) Representative super resolution images of cardiomyocytes isolated from ZSF1 obese rats treated with vehicle or VASHi are shown (scale bar=10μm) stained for detyrosinated-tubulin in gold or tyrosinated-tubulin in blue. Statistical significance was determined using two-way ANOVA with factors of genotype and treatment.* P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001. All data shown are mean ± SEM.

Properties of transverse and longitudinal stiffness were evaluated in cardiomyocytes isolated from lean and obese ZSF1 rats. Transverse stiffness was evaluated using nanoindentation, with elastic modulus measured at increasing nanoindentation velocities to determine viscoelastic properties (Fig. 5A-B). Cardiomyocytes isolated from obese rats had elevated stiffness at high indentation velocities, indicative of increased viscoelasticity. VASHi or TTL overexpression reduced cardiomyocyte viscoelasticity in both ZSF1-obese and ZSF1-lean rats compared to vehicle treatment (Fig. 5A to B). Quantification of elastic stiffness (Emin), stiffness at maximal velocity of indentation (Emax), and Emax-Emin (ΔE), an indicator of viscoelasticity, indicated increased viscoelasticity of vehicle treated obese rat cardiomyocytes compared to lean counterparts, which was reduced by VASHi treatment or TTL overexpression (Fig. 5C to E).

Figure 5. Ex vivo VASH Inhibition Improves ZSF1 Obese Rat Myocyte Stiffness at 30 Weeks of Age.

Figure 5.

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(A-B) Transverse stiffness (elastic modulus) is plotted as a function of nanoindentation velocity in (A) ZSF1-lean and (C) ZSF1-obese isolated cardiomyocytes (N = 5 lean rats, n = 19 myocytes treated with vehicle, 18 TTL, 10 VASHi; N = 6 obese rats, n = 29 myocytes treated with vehicle, 29 TTL, 15 VASHi). Vehicle treatment is shown in black, TTL treatment in pink, and SVC-01 in blue. (C-E) Quantification is shown of (C) Emin (elastic stiffness), (D) Emax (stiffness at maximal velocity of indentation) and (E) ΔE (indicator of viscoelasticity) in ZSF1 lean and ZSF1 obese cardiomyocytes (N = 5 lean rats, n = 19 myocytes treated with vehicle, 18 TTL, 10 VASHi; N = 6 obese rats, n = 29 myocytes treated with vehicle, 29 TTL, 15 VASHi). (F) A schematic to demonstrate how diastolic stiffness, steady state stiffness, and viscoelastic (stress) relaxation are determined in the diastolic stretch protocol. (G) Average trace of passive force versus time upon diastolic stretch protocol. (H) Zoom-in of myocyte passive force during the diastolic stretch. (I) Average passive force plotted against sarcomere length during diastolic stretch, with single exponential fit with goodness of fit (r2) and stiffness coefficient (b). (J) Diastolic stiffness (elastic modulus at the initial time of peak strain) upon a 10%, 200ms strain along the long axis of an isolated cardiomyocyte. (K) Steady state stiffness at end of strain and hold. (L) Viscoelastic stress relaxation in ZSF1-lean and ZSF1-obese cardiomyocytes treated with vehicle or VASH inhibitor (N = 4 lean rats, n = 10 myocytes treated with vehicle, 8 VASHi; N = 5 obese rats, n = 12 myocytes treated with vehicle, 11 with VASHi). For C-E and J-L, statistical significance was determined using two-way ANOVA with factors of phenotype and treatment. P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001. All data shown are mean ± SEM.

Longitudinal stiffness was evaluated using single myocyte stretch. In this experiment, myocytes were adhered to MyoTak coated laser-etched glass rods, one attached to a length controller and the other a force transducer. Force was measured while the myocyte was stretched from a resting to end diastolic sarcomere length at a rate consistent with diastolic filling (diastolic stiffness). This stretch was then held to evaluate stress relaxation (viscoelasticity) and steady state (elastic) stiffness, followed by return to resting length (see schematic and average traces in Fig. 5F to H). Vehicle treated obese rat cardiomyocytes had increased diastolic stiffness compared to vehicle treated lean cardiomyocytes, as evident by the increased slope of the force vs. sarcomere length relationship during stretch (Fig. 5I) and quantification of peak stiffness (Fig. 5J). Diastolic stiffness was normalized by VASHi treatment (Fig. 5J), with similar results observed for steady state stiffness and stress relaxation (Fig. 5K and L). Taken together, these results demonstrated that cardiomyocytes from ZSF1-obese rats had increased stiffness and viscoelasticity compared with lean ZSF controls, and that increased cardiomyocytes stiffness could be normalized by VASHi treatment.

Acute in vivo VASH Inhibition Improves Early Diastolic Relaxation

We proceeded to test the more potent compound (SVC-02) in vivo (Fig. 6A) in 30-week-old animals. The dosing schedule (two doses of 6.6mg/kg 24 hours apart) was based on the timing study performed in Swiss Webster mice, where peak reduction in detyrosination was observed at 8 hours post-treatment (fig. S3I and J). Western blot analysis validated that obese rats had robustly elevated detyrosinated tubulin compared to WKY and lean controls at this time point, and that intravenous delivery of SVC-02 significantly reduced detyrosinated tubulin in the myocardium (ob VASHi vs. ob veh: P=0.0005) (Fig. 6B and C). This decrease was also visualized by super resolution immunofluorescence imaging of fixed LV tissue sections from obese animals treated with vehicle controls or VASHi (Fig. 6D and E). A technical control showing specificity of the microtubule staining is shown in fig. S6. VASHi did not alter total microtubule density in obese animals, but induced a reduction in the density of detyrosinated microtubules compared to vehicle treatment (Fig. 6E).

Figure 6. Acute VASH Inhibition Improves ZSF1 Obese Rat Relaxation in vivo at 30 Weeks of Age.

Figure 6.

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(A) Timeline showing treatment protocol and assessments: at 30 weeks of age, ZSF1 obese, ZSF1 lean, and WKY rats received two treatments of SVC-02 (VASHi) 24 hours apart and assessed as indicated. (B) Western blot showing detyrosinated tubulin and GAPDH from WKY, ZSF1 lean, and ZSF1 obese LV tissue collected after two doses of VASHi. (C) Quantification of protein abundance for detyrosinated tubulin normalized to GAPDH from blots represented in panel B (N = 3 rats per group). (D) Immunofluorescence images show the abundance of detyrosinated-tubulin (magenta) and α-tubulin (yellow) with wheat germ agglutin (WGA) (green) in ZSF1 obese rats treated with vehicle (left) or ZSF1 obese rats treated with VASHi (right) (scale bar=10μm). (E) Quantification is shown for total microtubule density (left) and detyrosinated microtubule density (right) from the images represented in panel D (N= 3 rats per group). (F) Body weight, (G) cardiac hypertrophy, (H) mean arterial pressure, and (I) LV ejection fraction are shown (N = 5 9 rats per group, black is vehicle, blue is VASHi). (J) Tau, (K) MV deceleration time, (L) LVEDP, and (M) LA area are shown as metrics of diastolic function (N = 5 9 rats per group). As noted in panel A, all echocardiography measurements (K, M) were collected 8 hours after the first dose of VASHi, while hemodynamic (H, I, J, L) and descriptive (F, G) parameters were assessed 8 hours after the second dose. Statistical significance was determined using (C, F-M) two-way ANOVA with factors of phenotype and treatment or (E) one-way ANOVA. P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001. All data shown are mean ± SEM.

Though acute VASHi had no effect on body weight, heart weight, blood pressure, or EF (Fig. 6F to I), parameters of diastolic relaxation - tau and MV deceleration time - decreased significantly in ZSF1 obese animals treated with VASHi compared to vehicle-treated animals (tau – ob VASHi vs. ob veh: P=0.01; MV decel time – ob VASHi vs. ob veh: P=0.01) (Fig. 6J and K). Reductions in LVEDP with VASHi in obese rats did not achieve statistical significance when assessed using two-way ANOVA with both group and treatment taken into consideration (Fig. 6L), but a treatment effect within the obese group is apparent upon focused assessment (fig. S7A to C). Left atrial area was larger in obese ZSF1 rats with a non-significant trend toward normalization upon acute VASHi treatment (Fig. 6M). We also evaluated the impact of VASHi on circulating inflammatory markers in lean and obese animals, and found no difference in serum concentrations of 18 different analytes (table S3). Vehicle treated obese animals had significantly higher concentration of VEGF (P=0.04) and RANTES (P=0.03) than WKY animals. Additional echocardiography and invasive hemodynamic parameters are presented in table S4.

VASH Inhibition Improves Relaxation in Isolated Human Cardiomyocytes

Cardiomyocytes were isolated from failing (N=4) and non-failing (N=3) human hearts, then treated with vehicle or VASHi to evaluate bioactivity of the small molecule compound in primary human cardiomyocytes. Patient demographics are detailed in Table 1. Compiled traces from all non-failing and failing cardiomyocytes are shown in Figure 7A-B. Relaxation velocity was significantly increased with VASHi in both non-failing (P=0.001) and failing cardiomyocytes (P=0.03) (Fig. 7C). VASHi shortened the time to relxation at 10%, 50%, and 90% of baseline, paralleling observations in isolated cardiomyocytes and in vivo with obese rats (Fig. 7D to F). VASHi did not change fractional shortening, contraction velocity or resting sarcomere length in failing human cardiomyocytes (Fig. 7G to I).

Table 1. Patient Demographics.

Important demographics for explanted human hearts included in the VASH inhibitor testing. Statistical significance was assessed using unpaired t-tests. All data shown are mean ± SEM.

NF HF
N = 3 N = 4 P-value
Age 59.0 ± 6.4 50.0 ± 3.0 0.381
Sex 3 Female 4 Male
Mechanical Support - 1 ECMO
1 ECMO + Impella
2 Impella
Etiology - 1 Non-ischemic
1 Ischemic
2 Sarcoid
LV Ejection Fraction (%) 49.3 ± 2.3 13.1 ± 2.6 0.001
LV Mass (g) 185.0 ± 7.6 246.5 ± 10.8 0.031
Body Mass Index (kg/m2) 25.9 ± 1.3 25.0 ± 10.8 0.637
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Figure 7. Relaxation Is Improved in Isolated Failing Human Cardiomyocytes with VASH Inhibition.

Figure 7.

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(A-B) Average sarcomere shortening is shown for vehicle treated (black) and SVC-02 (VASHi) treated (blue) cardiomyocytes isolated from non-failing (NF) and (B) failing (HF) human hearts. (C-F) Shown is (C) relaxation velocity, and time to (D) 10%, (E) 50%, and (F) 90% relaxation. (G) Fractional shortening, (H) contraction velocity, and (I) resting sarcomere length are shown. N = 4 patients with HF, 3 patients with NF; n = 43 – 64 cardiomyocytes per group. Statistical significance was determined using two-way ANOVA with factors of phenotype and treatment. P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001. All data shown are mean ± SEM.

DISCUSSION

Evidence of diastolic dysfunction is clinically used to define the presence of HFpEF, however, there is uncertainty about targetable mechanisms contributing to diastolic dysfunction in humans and animal models of HFpEF. In this context, the present studies employ the ZSF1 obese rat model in which persistent hypertension, obesity, and diabetes conspire to induce progressive cardiac remodeling, impaired functional reserve, and relaxation defects resembling a well-defined subpopulation of patients with HFpEF (26). We observed that ZSF1 obese rats manifest left ventricular remodeling and impaired functional reserve, as observed in patients with HFpEF. These in vivo abnormalities in ZSF1 rats are associated with substantial increases in the ex vivo stiffness, viscoelasticity and relaxation times of isolated cardiomyocytes from ZSF1 obese rats compared with ZSF1 lean controls. In addition, increased expression of the tubulin carboxypeptidase VASH1 and improved cellular relaxation with a VASH inhibitor support a functional role for increased detyrosination of the microtubule network in mediating increased cardiomyocyte stiffness and viscoelasticity in ZSF1 obese rats. Moreover, improved early diastolic relaxation in ZSF1 obese rats following VASH inhibition suggest that increased microtubule network detyrosination is contributing to diastolic function defects in experimental HFpEF. Finally, improved cellular relaxation responses in isolated failing human cardiomyocytes demonstrates target engagement beyond the rodent model.

The ZSF1 rat has been used by multiple groups to study different interventions for a variety of pathologies, since these animals develop type 2 diabetes (14), metabolic syndrome (15, 16), diabetic nephropathy (17, 18), skeletal muscle dysfunction (19), and hypertension (27) in addition to the cardiac phenotype. Our echocardiography and invasive hemodynamic findings are in line with previous reports describing the development of diastolic dysfunction without impairments in systolic function. Although dP/dtmax is increased in ZSF1 obese rats, this is likely due to increased left ventricular end-diastolic pressure and aortic diastolic pressure as previously demonstrated rather than indicating an intrinsic increase in LV systolic function (28). Whereas extracardiac mechanisms, including skeletal muscle dysfunction and reduced diaphragm flexibility (29) (19) have clearly been shown to play a role, our demonstration of a heart-rate dependent increase in LVEDP suggests diastolic dysfunction. When comparing our phenotyping of the obese rat to clinical phenogroups of patients with HFpEF, our data and the predisposing features of the ZSF1 rats align most closely with phenogroup 3 described by Cohen et al., which was comprised of patients with obesity, diabetes, hypertension, abnormal metabolism, and liver and kidney dysfunction (26). Compared to the other two phenogroups, these patients had the highest plasma concentrations inflammatory biomarkers, concentric cardiac hypertrophy and E/e’ ratios indicative of diastolic dysfunction.

Our findings in isolated cardiomyocytes extend prior studies implicating intracellular contributors to abnormal diastolic function in HFpEF (30, 31). Though extracellular fibrosis (3) or amyloidosis (4) can increase myocardial stiffness in some patients with HFpEF, intracellular abnormalities of calcium handling (32) and alterations in titin isoform expression and phosphorylation within cardiomyocytes (33) have also been implicated as contributors to increased myocardial stiffness. In this context, demonstration of increased Vash1 expression, increased α-tubulin detyrosination and reductions in cardiomyocyte stiffness and viscoelasticity by targeting detyrosination with VASH inhibition in ZSF1 obese rats indicates that increased stabilization of the cardiomyocyte MTN is another intracellular contributor to diastolic function in HFpEF. Our lean rats also exhibited increased detyrosinated tubulin compared to WKY animals, suggesting these animals may develop some degree of microtubule driven stiffness as a result of their hypertensive phenotype. These findings in the ZSF1 obese rat add to a growing body of literature demonstrating that upregulation of tubulin carboxypeptidases (VASH1/2-SVBP) stabilizes the microtubule network in cardiomyopathies and (to a lesser extent) pressure overload hypertrophy (7, 24, 25).

Responses to acute in vivo VASH inhibition demonstrate that mitigating α-tubulin detyrosination improves early diastolic function even in the presence of other potential contributors to stiffness and relaxation. Moreover, the improvements of early myocardial relaxation with acute in vivo VASHi paralleled the improvements in isolated myocyte relaxation with ex vivo VASHi.

Finally, to extend our findings from an animal to primary human tissue, we treated cardiomyocytes isolated from failing and non-failing human hearts with VASHi. We have previously reported that both parthenolide (which indirectly reduces microtubule detyrosination) (34) and colchicine (which destabilizes microtubules) improved relaxation of failing human myocytes. However, both of these drugs have undesirable qualities that limit their use as a microtubule targeted therapy (7, 11, 35, 36). By designing a specific, targeted, and potent small molecule compound, we overcome these disadvantages of older treatments with a new compound that may favor clinical translation.

Our study has several limitations. We only employed short-term VASHi and findings could be amplified, diminished, or different with sustained VASHi. Based on time course data (Fig. 1A to B) that revealed earlier than anticipated elevations in detyrosination and features of HFpEF in ZSF1 obese rats, earlier and chronic intervention studies should also be pursued. All failing human hearts included in this study had reduced ejection fraction. Patients with HFpEF rarely receive heart transplants, so we tested VASHi in the explanted human hearts we received from patients with HFrEF. We exclusively used male animals in this study but acknowledge that studying both sexes is important. A previous study described that female obese rats have a similar cardiac phenotype as males, but do not develop hyperglycemia (20). Both male and female obese ZSF1 rats develop cardiac dysfunction in obesity-induced heart failure with preserved ejection fraction (20).

The present studies identify increased α-tubulin detyrosination as a contributor to increased cardiomyocyte stiffness and impaired relaxation. We demonstrate that a new small molecule VASH inhibitor reverses increased tubulin detyrosination and speeds relaxation, supporting a potential therapeutic role in HFpEF.

MATERIALS AND METHODS

Study design

The objective of this study was to test if VASH-dependent MTN detyrosination contributed to impaired diastolic function in a model of HFpEF (obese ZSF1 rat) both ex vivo and in vivo using a newly developed vasohibin inhibitor (VASHi). Sample sizes were determined using power analyses based on effect size observed during preliminary experiments. All animals were randomly selected for VASHi or vehicle treatment and investigators were blinded from data collection through analysis. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Perelman School of Medicine of the University of Pennsylvania under protocol #807166. Procurement of human myocardial tissue was performed under protocols and ethical regulations approved by Institutional Review Boards at the University of Pennsylvania and the Gift-of-Life Donor Program (Pennsylvania, USA). ZSF1 obese, lean, Wistar Kyoto (WKY), and Sprague Dawley rats were purchased from Charles River Laboratories. Only male rats were used in this study.

Vasohibin Inhibition

For experiments reporting results from isolated rodent cardiomyocytes, an earlier version of the vasohibin inhibitor (VASHi), SVC-01, was used. For experiments treating ZSF1 obese, lean, and Wistar Kyoto rats in vivo and isolated human cardiomyocytes, a newer version of the VASHi, SVC-02, was used. We have outlined which experiments used SVC-01 and SVC-02 in table S5.

Human Myocardial Tissue Procurement

Failing human hearts were procured at the time of orthotropic heart transplantation at the Hospital of the University of Pennsylvania following prospective informed consent from all participants. Non-failing hearts were obtained at the time of organ donation from cadaveric donors. Consent for use of donors’ heart tissue for research was obtained from next-of-kin. In all cases, hearts were arrested in situ using ice-cold cardioplegia solution and transported to the laboratory on wet ice.

Statistical analysis

Statistical analysis was performed using GraphPad 7.05 (GraphPad Software) and Origin Pro 2019 (OriginLab Corporation). Data shown are means ± standard error or individual data with bar graphs representing means and standard error. For experiments comparing 2 groups, unpaired t-tests were used. For experiments comparing 3 groups, one-way ANOVA was used. For experiments comparing 6 groups, two-way ANOVA was used. The statistical test used for each experiment has been included in the appropriate figure legend along with the sample size/replicates included. N represents the number of rats or human hearts and n is the number of single cells used during analysis.

Supplementary Material

Supplementary Materials
Data File S1
MDAR Checklist

ACKNOWLEDGEMENTS

The echocardiography was performed by the Rodent Cardiovascular Phenotyping Core (RRID: SCR_022419) at the University of Pennsylvania supported by the Penn Cardiovascular Institute and NIH S10OD016393. We are grateful to Joanna Griffin for performing all echocardiography included in this manuscript. The authors thank Yangzhu Du, Honghong Sun, and Nina Luning Prak of the Human Immunology Core at the Perelman School of Medicine at the University of Pennsylvania (HIC RRID: SCR_022380) for assistance with the Rat serum Luminex assay. The HIC is supported in part by NIH P30 AI045008 and P30 CA016520. The authors acknowledge the assistance of the Gift of Life Donor Program, Philadelphia, PA, which helped provide non-failing heart tissue from transplant rejected donor hearts for this research. We acknowledge the imaging facility MRI, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04) «Investments for the future».

Funding:

This research was funded by National Heart Lung and Blood Institute of National Institutes of Health R01-HL149891–01 to KBM and BLP, National Heart Lung and Blood Institute of National Institutes of Health R01- HL133080 to BLP, National Heart Lung and Blood Institute of National Institutes of Health T32 HL007843 to DME and BWL, BPI France DOS0166797/00 to SvdL and KH, Leducq Foundation award TNE ID#: 673168 to KBM and BLP, AHA CDA 856504 to MAC, sponsored research agreement with MT-act SAS to KBM and BLP, Center for Engineering Mechanobiology through a grant from the National Science Foundation’s Science and Technology program: 15–48571 to BLP.

Footnotes

Competing interests: Benjamin L. Prosser, Kenneth B. Margulies, Khaled Hached, and Siem van der Laan are co inventors for “International patent application No. EP2024/054701 for Specific VASH Compounds (SVC) Inhibitors for Heart Diseases”. Guillaume Marcellin, Yoann Lannay are employees and Khaled Hached and Siem van der Laan are employees and shareholders of MT-act.

Data and materials availability:

All data associated with this study are present in the paper or supplementary materials. The vasohibin inhibitors used in the study were synthesized by MT-act and provided to investigators at the University of Pennsylvania through a materials transfer agreement.

REFERENCES

  • 1.Heidenreich PA, Bozkurt B, Aguilar D, Allen LA, Byun JJ, Colvin MM, Deswal A, Drazner MH, Dunlay SM, Evers LR, Fang JC, Fedson SE, Fonarow GC, Hayek SS, Hernandez AF, Khazanie P, Kittleson MM, Lee CS, Link MS, Milano CA, Nnacheta LC, Sandhu AT, Stevenson LW, Vardeny O, Vest AR, Yancy CW, 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure: Executive Summary: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. J Am Coll Cardiol 79, 1757–1780 (2022). [DOI] [PubMed] [Google Scholar]
  • 2.Kraigher-Krainer E, Shah AM, Gupta DK, Santos A, Claggett B, Pieske B, Zile MR, Voors AA, Lefkowitz MP, Packer M, McMurray JJ, Solomon SD, Investigators P, Impaired systolic function by strain imaging in heart failure with preserved ejection fraction. J Am Coll Cardiol 63, 447–456 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zile MR, Baicu CF, Ikonomidis JS, Stroud RE, Nietert PJ, Bradshaw AD, Slater R, Palmer BM, Van Buren P, Meyer M, Redfield MM, Bull DA, Granzier HL, LeWinter MM, Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin. Circulation 131, 1247–1259 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hahn VS, Yanek LR, Vaishnav J, Ying W, Vaidya D, Lee YZJ, Riley SJ, Subramanya V, Brown EE, Hopkins CD, Ononogbu S, Perzel Mandell K, Halushka MK, Steenbergen C Jr., Rosenberg AZ, Tedford RJ, Judge DP, Shah SJ, Russell SD, Kass DA, Sharma K, Endomyocardial Biopsy Characterization of Heart Failure With Preserved Ejection Fraction and Prevalence of Cardiac Amyloidosis. JACC Heart Fail 8, 712–724 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Loescher CM, Hobbach AJ, Linke WA, Titin (TTN): from molecule to modifications, mechanics, and medical significance. Cardiovasc Res 118, 2903–2918 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Robison P, Caporizzo MA, Ahmadzadeh H, Bogush AI, Chen CY, Margulies KB, Shenoy VB, Prosser BL, Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes. Science 352, aaf0659 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chen CY, Caporizzo MA, Bedi K, Vite A, Bogush AI, Robison P, Heffler JG, Salomon AK, Kelly NA, Babu A, Morley MP, Margulies KB, Prosser BL, Suppression of detyrosinated microtubules improves cardiomyocyte function in human heart failure. Nat Med 24, 1225–1233 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Sanyal C, Pietsch N, Ramirez Rios S, Peris L, Carrier L, Moutin MJ, The detyrosination/re-tyrosination cycle of tubulin and its role and dysfunction in neurons and cardiomyocytes. Semin Cell Dev Biol 137, 46–62 (2023). [DOI] [PubMed] [Google Scholar]
  • 9.Salomon AK, Phyo SA, Okami N, Heffler J, Robison P, Bogush AI, Prosser BL, Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte. Basic Res Cardiol 117, 53 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Cheng G, Zile MR, Takahashi M, Baicu CF, Bonnema DD, Cabral F, Menick DR, G. t. Cooper, A direct test of the hypothesis that increased microtubule network density contributes to contractile dysfunction of the hypertrophied heart. Am J Physiol Heart Circ Physiol 294, H2231–2241 (2008). [DOI] [PubMed] [Google Scholar]
  • 11.Koide M, Hamawaki M, Narishige T, Sato H, Nemoto S, DeFreyte G, Zile MR, Cooper GI, Carabello BA, Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy. Circulation 102, 1045–1052 (2000). [DOI] [PubMed] [Google Scholar]
  • 12.Tsutsui H, Ishihara K, G. t. Cooper, Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science 260, 682–687 (1993). [DOI] [PubMed] [Google Scholar]
  • 13.Aillaud C, Bosc C, Peris L, Bosson A, Heemeryck P, Van Dijk J, Le Friec J, Boulan B, Vossier F, Sanman LE, Syed S, Amara N, Coute Y, Lafanechere L, Denarier E, Delphin C, Pelletier L, Humbert S, Bogyo M, Andrieux A, Rogowski K, Moutin MJ, Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science 358, 1448–1453 (2017). [DOI] [PubMed] [Google Scholar]
  • 14.Lesniewski LA, Donato AJ, Behnke BJ, Woodman CR, Laughlin MH, Ray CA, Delp MD, Decreased NO signaling leads to enhanced vasoconstrictor responsiveness in skeletal muscle arterioles of the ZDF rat prior to overt diabetes and hypertension. Am J Physiol Heart Circ Physiol 294, H1840–1850 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Park SH, Farooq MA, Gaertner S, Bruckert C, Qureshi AW, Lee HH, Benrahla D, Pollet B, Stephan D, Ohlmann P, Lessinger JM, Mayoux E, Auger C, Morel O, Schini-Kerth VB, Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat. Cardiovasc Diabetol 19, 19 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Stolina M, Luo X, Dwyer D, Han CY, Chen R, Zhang Y, Xiong Y, Chen Y, Yin J, Shkumatov A, Ason B, Hale C, Veniant MM, The evolving systemic biomarker milieu in obese ZSF1 rat model of human cardiometabolic syndrome: Characterization of the model and cardioprotective effect of GDF15. PLoS One 15, e0231234 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bilan VP, Salah EM, Bastacky S, Jones HB, Mayers RM, Zinker B, Poucher SM, Tofovic SP, Diabetic nephropathy and long-term treatment effects of rosiglitazone and enalapril in obese ZSF1 rats. J Endocrinol 210, 293–308 (2011). [DOI] [PubMed] [Google Scholar]
  • 18.Boustany-Kari CM, Harrison PC, Chen H, Lincoln KA, Qian HS, Clifford H, Wang H, Zhang X, Gueneva-Boucheva K, Bosanac T, Wong D, Fryer RM, Richman JG, Sarko C, Pullen SS, A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat. J Pharmacol Exp Ther 356, 712–719 (2016). [DOI] [PubMed] [Google Scholar]
  • 19.Schauer A, Draskowski R, Jannasch A, Kirchhoff V, Goto K, Mannel A, Barthel P, Augstein A, Winzer E, Tugtekin M, Labeit S, Linke A, Adams V, ZSF1 rat as animal model for HFpEF: Development of reduced diastolic function and skeletal muscle dysfunction. ESC Heart Fail 7, 2123–2134 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Nguyen ITN, Brandt MM, van de Wouw J, van Drie RWA, Wesseling M, Cramer MJ, de Jager SCA, Merkus D, Duncker DJ, Cheng C, Joles JA, Verhaar MC, Both male and female obese ZSF1 rats develop cardiac dysfunction in obesity-induced heart failure with preserved ejection fraction. PLoS One 15, e0232399 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.van der Laan S, Leveque MF, Marcellin G, Vezenkov L, Lannay Y, Dubra G, Bompard G, Ovejero S, Urbach S, Burgess A, Amblard M, Sterkers Y, Bastien P, Rogowski K, Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination. Cell Rep 29, 4159–4171 e4156 (2019). [DOI] [PubMed] [Google Scholar]
  • 22.Nieuwenhuis J, Adamopoulos A, Bleijerveld OB, Mazouzi A, Stickel E, Celie P, Altelaar M, Knipscheer P, Perrakis A, Blomen VA, Brummelkamp TR, Vasohibins encode tubulin detyrosinating activity. Science 358, 1453–1456 (2017). [DOI] [PubMed] [Google Scholar]
  • 23.Schuldt M, Pei J, Harakalova M, Dorsch LM, Schlossarek S, Mokry M, Knol JC, Pham TV, Schelfhorst T, Piersma SR, Dos Remedios C, Dalinghaus M, Michels M, Asselbergs FW, Moutin MJ, Carrier L, Jimenez CR, van der Velden J, Kuster DWD, Proteomic and Functional Studies Reveal Detyrosinated Tubulin as Treatment Target in Sarcomere Mutation-Induced Hypertrophic Cardiomyopathy. Circ Heart Fail 14, e007022 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Chen CY, Salomon AK, Caporizzo MA, Curry S, Kelly NA, Bedi K, Bogush AI, Kramer E, Schlossarek S, Janiak P, Moutin MJ, Carrier L, Margulies KB, Prosser BL, Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes. Circ Res 127, e14–e27 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yu X, Chen X, Amrute-Nayak M, Allgeyer E, Zhao A, Chenoweth H, Clement M, Harrison J, Doreth C, Sirinakis G, Krieg T, Zhou H, Huang H, Tokuraku K, Johnston DS, Mallat Z, Li X, MARK4 controls ischaemic heart failure through microtubule detyrosination. Nature 594, 560–565 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Cohen JB, Schrauben SJ, Zhao L, Basso MD, Cvijic ME, Li Z, Yarde M, Wang Z, Bhattacharya PT, Chirinos DA, Prenner S, Zamani P, Seiffert DA, Car BD, Gordon DA, Margulies K, Cappola T, Chirinos JA, Clinical Phenogroups in Heart Failure With Preserved Ejection Fraction: Detailed Phenotypes, Prognosis, and Response to Spironolactone. JACC Heart Fail 8, 172–184 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Nguyen ITN, Cramer MJ, Joles JA, Verhaar MC, Renal injury in relation to obesity and the additive effect of hypertension in female and male obese and lean ZSF1 rats. Am J Physiol Renal Physiol 325, F73–F86 (2023). [DOI] [PubMed] [Google Scholar]
  • 28.Wallace AG, Skinner NS Jr., Mitchell JH, Hemodynamic determinants of the maximal rate of rise of left ventricular pressure. Am J Physiol 205, 30–36 (1963). [DOI] [PubMed] [Google Scholar]
  • 29.Bowen TS, Brauer D, Rolim NPL, Baekkerud FH, Kricke A, Ormbostad Berre AM, Fischer T, Linke A, da Silva GJ, Wisloff U, Adams V, Exercise Training Reveals Inflexibility of the Diaphragm in an Animal Model of Patients With Obesity-Driven Heart Failure With a Preserved Ejection Fraction. J Am Heart Assoc 6, (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Paulus WJ, Zile MR, From Systemic Inflammation to Myocardial Fibrosis: The Heart Failure With Preserved Ejection Fraction Paradigm Revisited. Circ Res 128, 1451–1467 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Pfeffer MA, Shah AM, Borlaug BA, Heart Failure With Preserved Ejection Fraction In Perspective. Circ Res 124, 1598–1617 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Frisk M, Le C, Shen X, Roe AT, Hou Y, Manfra O, Silva GJJ, van Hout I, Norden ES, Aronsen JM, Laasmaa M, Espe EKS, Zouein FA, Lambert RR, Dahl CP, Sjaastad I, Lunde IG, Coffey S, Cataliotti A, Gullestad L, Tonnessen T, Jones PP, Altara R, Louch WE, Etiology-Dependent Impairment of Diastolic Cardiomyocyte Calcium Homeostasis in Heart Failure With Preserved Ejection Fraction. J Am Coll Cardiol 77, 405–419 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.van Heerebeek L, Franssen CP, Hamdani N, Verheugt FW, Somsen GA, Paulus WJ, Molecular and cellular basis for diastolic dysfunction. Curr Heart Fail Rep 9, 293–302 (2012). [DOI] [PubMed] [Google Scholar]
  • 34.Hotta T, Haynes SE, Blasius TL, Gebbie M, Eberhardt EL, Sept D, Cianfrocco M, Verhey KJ, Nesvizhskii AI, Ohi R, Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin. Curr Biol 31, 900–907 e906 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Tsutsui H, Ishibashi Y, Takahashi M, Namba T, Tagawa H, Imanaka-Yoshida K, Takeshita A, Chronic colchicine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats. J Mol Cell Cardiol 31, 1203–1213 (1999). [DOI] [PubMed] [Google Scholar]
  • 36.Scopacasa BS, Teixeira VP, Franchini KG, Colchicine attenuates left ventricular hypertrophy but preserves cardiac function of aortic-constricted rats. J Appl Physiol (1985) 94, 1627–1633 (2003). [DOI] [PubMed] [Google Scholar]
  • 37.Zacchigna S, Paldino A, Falcao-Pires I, Daskalopoulos EP, Dal Ferro M, Vodret S, Lesizza P, Cannata A, Miranda-Silva D, Lourenco AP, Pinamonti B, Sinagra G, Weinberger F, Eschenhagen T, Carrier L, Kehat I, Tocchetti CG, Russo M, Ghigo A, Cimino J, Hirsch E, Dawson D, Ciccarelli M, Oliveti M, Linke WA, Cuijpers I, Heymans S, Hamdani N, de Boer M, Duncker DJ, Kuster D, van der Velden J, Beauloye C, Bertrand L, Mayr M, Giacca M, Leuschner F, Backs J, Thum T, Towards standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on Myocardial Function. Cardiovasc Res 117, 43–59 (2021). [DOI] [PubMed] [Google Scholar]
  • 38.Altshuler PJ, Schiazza AR, Luo L, Helmers MR, Chhay B, Han JJ, Hu R, Herbst DA, Tsourkas A, Cheng Z, Atluri P, Superoxide Dismutase-Loaded Nanoparticles Attenuate Myocardial Ischemia-Reperfusion Injury and Protect Against Chronic Adverse Ventricular Remodeling. Adv Ther (Weinh) 4, (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Caporizzo MA, Chen CY, Salomon AK, Margulies KB, Prosser BL, Microtubules Provide a Viscoelastic Resistance to Myocyte Motion. Biophys J 115, 1796–1807 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Vite A, Caporizzo MA, Corbin EA, Brandimarto J, McAfee Q, Livingston CE, Prosser BL, Margulies KB, Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms. Basic Res Cardiol 117, 41 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Scarborough EA, Uchida K, Vogel M, Erlitzki N, Iyer M, Phyo SA, Bogush A, Kehat I, Prosser BL, Microtubules orchestrate local translation to enable cardiac growth. Nat Commun 12, 1547 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Benoist L, Chadet S, Genet T, Lefort C, Heraud A, Danila MD, Muntean DM, Baron C, Angoulvant D, Babuty D, Bourguignon T, Ivanes F, Stimulation of P2Y11 receptor protects human cardiomyocytes against Hypoxia/Reoxygenation injury and involves PKCepsilon signaling pathway. Sci Rep 9, 11613 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Caporizzo MA, Chen CY, Bedi K, Margulies KB, Prosser BL, Microtubules Increase Diastolic Stiffness in Failing Human Cardiomyocytes and Myocardium. Circulation 141, 902–915 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
NIHMS2107089-supplement-Supplementary_Materials.docx (2MB, docx)
Data File S1
NIHMS2107089-supplement-Data_File_S1.xlsx (61.7KB, xlsx)
MDAR Checklist
NIHMS2107089-supplement-MDAR_Checklist.pdf (454.5KB, pdf)

Data Availability Statement

All data associated with this study are present in the paper or supplementary materials. The vasohibin inhibitors used in the study were synthesized by MT-act and provided to investigators at the University of Pennsylvania through a materials transfer agreement.

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