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Pressure overload-induced cardiac hypertrophy, such as that caused by hypertension, is a key risk factor for heart failure. However, the underlying molecular mechanisms remain largely unknown. We previously reported that the valosin-containing protein (VCP), an ATPase-associated protein newly identified in the heart, acts as a significant mediator of cardiac protection against pressure overload-induced pathological cardiac hypertrophy. Still, the underlying molecular basis for the protection is unclear. This study used a cardiac-specific VCP transgenic mouse model to understand the transcriptomic alterations induced by VCP under the cardiac stress caused by pressure overload. Using RNA sequencing and comprehensive bioinformatic analysis, we found that overexpression of the VCP in the heart was able to normalize the pressure overload-stimulated hypertrophic signals by activating G protein-coupled receptors, particularly, the olfactory receptor family, and inhibiting the transcription factor controlling cell proliferation and differentiation. Moreover, VCP overexpression restored pro-survival signaling through regulating alternative splicing alterations of mitochondrial genes. Together, our study revealed a novel molecular regulation mediated by VCP under pressure overload that may bring new insight into the mechanisms involved in protecting against hypertensive heart failure.

Chronic hypertension is a key risk factor for heart failure. However, the underlying molecular mechanisms are not fully understood. Our previous studies found that the valosin-containing protein (VCP), an ATPase-associated protein, was significantly decreased in the hypertensive heart tissues. In this study, we tested the hypothesis that restoration of VCP protected the heart against pressure overload-induced heart failure. With a cardiac-specific transgenic (TG) mouse model, we showed that a moderate increase of VCP was able to attenuate chronic pressure overload-induced maladaptive cardiac hypertrophy and dysfunction. RNA sequencing and a comprehensive bioinformatic analysis further demonstrated that overexpression of VCP in the heart normalized the pressure overload-stimulated hypertrophic signals and repressed the stress-induced inflammatory response. In addition, VCP overexpression promoted cell survival by enhancing the mitochondria resistance to the oxidative stress via activating the Rictor-mediated-gene networks. VCP was also found to be involved in the regulation of the alternative splicing and differential isoform expression for some genes that are related to ATP production and protein synthesis by interacting with long no-coding RNAs and histone deacetylases, indicating a novel epigenetic regulation of VCP in integrating coding and noncoding genomic network in the stressed heart. In summary, our study demonstrated that the rescuing of a deficient VCP in the heart could prevent pressure overload-induced heart failure by rectifying cardiac hypertrophic and inflammatory signaling and enhancing the cardiac resistance to oxidative stress, which brought in novel insights into the understanding of the mechanism of VCP in protecting patients from hypertensive heart failure., Graphical abstract Image 1, Highlights • Deficiency of VCP contributes to the pathogenesis of hypertensive heart failure. • Rescue of VCP prevents stress-induced cardiac remodeling and cell death. • VCP attenuates stress-induced inflammatory and hypertrophic signaling. • VCP promotes cardiac resistance to oxidative stress. • VCP mediates a novel epigenetic integrating regulation in the stressed heart.

Valosin-containing protein (VCP) was found to play a vital protective role against cardiac stresses. Genetic mutations of VCP are associated with human dilated cardiomyopathy. However, the essential role of VCP in the heart during the physiological condition remains unknown since the VCP knockout in mice is embryonically lethal. We generated a cardiac-specific dominant-negative VCP transgenic (DN-VCP TG) mouse to determine the effects of impaired VCP activity on the heart. Using echocardiography, we showed that cardiac-specific overexpression of DN-VCP induced a remarkable cardiac dilation and progressively declined cardiac function during the aging transition. Mechanistically, DN-VCP did not affect the endogenous VCP (EN-VCP) expression but significantly reduced cardiac ATPase activity in the DN-VCP TG mouse hearts, indicating a functional inhibition. DN-VCP significantly impaired the aging-related cytoplasmic/nuclear shuffling of EN-VCP and its co-factors in the heart tissues and interrupted the balance of the VCP-cofactors interaction between the activating co-factors, ubiquitin fusion degradation protein 1 (UFD-1)/nuclear protein localization protein 4 (NPL-4) complex, and its inhibiting co-factor P47, leading to the binding preference with the inhibitory co-factor, resulting in functional repression of VCP. This DN-VCP TG mouse provides a unique functional-inactivation model for investigating VCP in the heart in physiological and pathological conditions.

Background/Aims: Our previous studies demonstrated that intrinsic aortic smooth muscle cell (VSMC) stiffening plays a pivotal role in aortic stiffening in aging and hypertension. However, the underlying molecular mechanisms remain largely unknown. We here hypothesized that Rho kinase (ROCK) acts as a novel mediator that regulates intrinsic VSMC mechanical properties through the serum response factor (SRF) /myocardin pathway and consequently regulates aortic stiffness and blood pressure in hypertension. Methods: Four-month old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were studied. Aortic stiffness was measured by echography. Intrinsic mechanical properties of VSMCs were measured by atomic force microscopy (AFM) in vitro. Results: Compared to WKY rats, SHR showed a significant increase in aortic stiffness and blood pressure, which is accompanied by a remarkable cell stiffening and ROCK activation in thoracic aortic (TA) VSMCs. Theses alterations in SHR were abolished by Y-27632, a specific inhibitor of ROCK. Additionally, boosted filamentous/globular actin ratio was detected in TA VSMCs from SHR versus WKY rats, resulting in an up-regulation of SRF and myocardin expression and its downstream stiffness-associated genes including α-smooth muscle actin, SM22, smoothelin and myosin heavy chain 11. Reciprocally, these alterations in SHR TA VSMCs were also suppressed by Y-27632. Furthermore, a specific inhibitor of SRF/myocardin, CCG-100602, showed a similar effect to Y-27632 in SHR in both TA VSMCs stiffness in vitro and aorta wall stiffness in vivo. Conclusion: ROCK is a novel mediator modulating aortic VSMC stiffness through SRF/myocardin signaling which offers a therapeutic target to reduce aortic stiffening in hypertension.

Summary Hypertension‐induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin‐containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4‐month male spontaneously hypertensive rats (SHRs) vs. age‐matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild‐type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin‐containing protein VCP expression was also observed to be time‐ and dose‐dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac‐specific overexpression of VCP showed a significant repression in TAC‐induced LVH vs. litter‐matched WT controls upon 2‐week TAC. TAC‐induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway.

Stress-induced mitochondrial calcium (Ca 2+ ) overload is a trigger of cardiomyocyte death though the opening of mitochondrial permeability transition pore (mPTP) during cardiac ischemic injury. We previously found that the valosin-containing protein (VCP), an ATPase-associated protein, protects cardiomyocytes against stress-induced death and also inhibits mPTP opening in vitro. However, the underlying molecular mechanisms are not fully understood. Here, we tested our hypothesis that VCP protects cardiomyocytes against stress-induced mPTP opening through resisting excessive mitochondrial Ca 2+ uptake. By using a cardiac-specific transgenic (TG) mouse model in which VCP is overexpressed by 3.5 folds in the hearts, we found that, under the pathological extra-mitochondrial Ca 2+ overload (10-70μM), Ca 2+ entered into cardiac mitochondriawas much lessin VCP TG mice compared to little-matched WT mice, subsequently preventing mPTP opening and ATP depletion under the Ca 2+ challenge. Mechanistically, overexpression of VCP in the heart results in post-translational protein degradation of the mitochondrial Ca 2+ uptake protein 1 (MICU1), an activator of the mitochondria Ca 2+ uniporter (MCU) which is responsible for mitochondrial calcium uptake. Together, our results reveal a new regulatory role of VCP in cardiac mitochondrial Ca 2+ homeostasis and unlocks the potential mechanism by which VCP confers its cardioprotection.

Stress-induced mitochondrial calcium (Ca2+) overload is a key cellular toxic effectors and a trigger of cardiomyocyte death during cardiac ischemic injury through the opening of mitochondrial permeability transition pore (mPTP). We previously found that the valosin-containing protein (VCP), an ATPase-associated protein, protects cardiomyocytes against stress-induced death and also inhibits mPTP opening in vitro. However, the underlying molecular mechanisms are not fully understood. Here, we tested our hypothesis that VCP acts as a novel regulator of mitochondrial Ca2+ uptake proteins and resists cardiac mitochondrial Ca2+ overload by modulating mitochondrial Ca2+ homeostasis. By using a cardiac-specific transgenic (TG) mouse model in which VCP is overexpressed by 3.5 folds in the heart compared to the wild type (WT) mouse, we found that, under the pathological extra-mitochondrial Ca2+ overload, Ca2+ entry into cardiac mitochondria was reduced in VCP TG mice compared to their little-matched WT mice, subsequently preventing mPTP opening and ATP depletion under the Ca2+ challenge. Mechanistically, overexpression of VCP in the heart resulted in post-translational protein degradation of the mitochondrial Ca2+ uptake protein 1, an activator of the mitochondria Ca2+ uniporter that is responsible for mitochondrial calcium uptake. Together, our results reveal a new regulatory role of VCP in cardiac mitochondrial Ca2+ homeostasis and unlock the potential mechanism by which VCP confers its cardioprotection.

Increased aortic stiffness is a fundamental manifestation of hypertension. However, the molecular mechanisms involved remain largely unknown. We tested the hypothesis that abnormal intrinsic vascular smooth muscle cell (VSMC) mechanical properties in large arteries, but not in distal arteries, contribute to the pathogenesis of aortic stiffening in hypertension, mediated by the serum response factor (SRF)/myocardin signalling pathway.Four month old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Using atomic force microscopy, significant VSMC stiffening was observed in the large conducting aorta compared with the distal arteries in SHR (P 0.001), however, this regional variation was not observed in WKY rats (P 0.4). The increase of VSMC stiffness was accompanied by a parallel increase in the expression of SRF by 9.8-fold and of myocardin by 10.5-fold in thoracic aortic VSMCs from SHR vs. WKY rats, resulting in a significant increase of downstream stiffness-associated genes (all, P 0.01 vs. WKY). Inhibition of SRF/myocardin expression selectively attenuated aortic VSMC stiffening, and normalized downstream targets in VSMCs isolated from SHR but not from WKY rats. In vivo, 2 weeks of treatment with SRF/myocardin inhibitor delivered by subcutaneous osmotic minipump significantly reduced aortic stiffness and then blood pressure in SHR but not in WKY rats, although concomitant changes in aortic wall remodelling were not detected during this time frame.SRF/myocardin pathway acts as a pivotal mediator of aortic VSMC mechanical properties and plays a central role in the pathological aortic stiffening in hypertension. Attenuation of aortic VSMC stiffening by pharmacological inhibition of SRF/myocardin signalling presents a novel therapeutic strategy for the treatment of hypertension by targeting the cellular contributors to aortic stiffness.

Summary Aortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening.

Aims: We previously showed that the valosin containing protein (VCP), a member of ATPases associated protein, protects the heart against pressure overload induced cardiac hypertrophy and dysfunction in transgenic (TG) mice. The mechanisms remain unknown. We hypothesize that VCP regulated alteration in transcriptome contributes to its cardio-protection by targeting cardiac cell growth and survival. Methods and results: Cardiac-specific VCPTG mice and their litter-matched wild type (WT) mice were subjected to transverse aortic constriction (TAC) for 2 and 5 weeks and compared to the sham mice. By using echocardiography and hemodynamic analysis, cardiac hypertrophy and dysfunction were confirmed in 2 weeks- and 5 weeks- TAC models respectively in WT but not in VCPTG mice. Total RNA was extracted from left ventricular tissues and gene expression was determined by RNA-Seq transcriptome analysis. Upon 2 weeks TAC, 690 differentially expressed genes were identified between VCPTG and WT (Fold Change ≥ 1.5, P value ≤ 0.05). Among these genes, VCPTG TAC mice, compared to WT TAC mice, showed significant activation of the genes linked to transcriptional factors, Protein Kinase CGMP-Dependent Type I ( Prkg1 ) and Kruppel Like factor 15 ( Klf15 ), the known repressors of cardiac hypertrophy under stress. On the contrary, there is significant increase in cardiac hypertrophy associated genes in WT TAC mice, such as myosin light chain 7 ( Myl7 ), periostin ( Postn ) and tropomyosin beta chain ( Tpm2) , and in fetal gene natriuretic peptide A ( Nppa) , but these alterations were not observed in VCPTG TAC mice, which may contribute to repression of cardiac hypertrophy in VCPTG mice. In addition, pro-apoptosis genes, such as platelet factor 4 ( Pf4), Pleckstrin homology like domain A1 ( Phlda1 ) and Radical S-Adenosyl Methionine Domain Containing 2 ( Rsad2 ), are significantly downregulated continually in 2 weeks through 5 weeks TAC in VCPTG mice, but not in WT TAC, which may contribute to the protection of cell survival in VCPTG mice. Conclusion: Significant difference of gene regulation exists between VCPTG and WT in the heart under the pressure overload which may be the mechanism of VCP mediated cardiac protection.

Background: An increase in aortic stiffness is a fundamental component of hypertension . We previous showed that aortic vascular smooth muscle cells (VSMCs) play an causative role in aortic stiffening not only due to the increased intrinsic stiffness in VSMC itself but also due to its interaction with extracellular matrix (ECM). However, the molecular mechanism involved is unclear. Here, we test the hypothesis that the aortic VSMCs mediates pre- and post-translational regulation of Lysyl Oxidase (Lox)/β1intergrain pathway which may be one of the mechanisms of VSMCs-ECM remodeling in hypertension. Methods and Results: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured with an invasive catheter and Doppler imaging echocardiography, respectively. Compared to WKY, SHR showed a significant higher aortic pressure (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, ( p p p p Conclusion: The regulation of pre- and post-translational regulation of Lox/β1intergrain pathway in aortic VSMCs presents a novel mechanism of cell-ECM interaction, which may contribute to the increased aortic stiffness in SHR.

Aims: The pressure overload induced cardiac hypertrophy is a key risk factor for heart failure. However, the mechanism involved remains largely unknown. We previously showed that overexpression of valosin-containing protein (VCP) promotes cardiomyocytes survival in vitro . Here we tested the hypothesis that VCP protects heart against pressure overload-induced heart failure via promoting pro-survival signaling in cardiomyocyte in vivo . Methods and Results: VCP expression were found to be notably decreased in left ventricle (LV) tissues of dilated cardiomyopathy patients compared to healthy donor at both the mRNA and protein levels. A transgenic mouse (TG) with a cardiac specific overexpression of VCP was generated and compared to its litter-matched wild-type (WT). Pressure overload was induced by transverse aortic constriction (TAC) in mice for 5 weeks and sham control was included. Cardiac structure and function were measured by echocardiography and hemodynamic analysis. After 5 week TAC, compared to sham control, WT mice developed a dilated cardiac hypertrophy and cardiac dysfunction, reflected by a significant increase in LV chamber diameter, wall thickness and the ratio of LV weight/ tibia length, and a decrease in ejection fraction (71% vs 51%) as well as an increase in ratio of lung weight /tibia length, a marker of heart failure (all, Pin vivo and in isolated cardiomyocytes in vitro which was abolished by the deletion of Rictor, indicating an activation of mammalian target of rapamycin complex 2 (mTORC2) in cardiomyocytes. Conclusion: Overexpression of VCP prevents TAC-induced cardiac remodeling and heart failure via a pro-survival mechanism involving mTORC2 signaling pathway.

The valosin-containing protein (VCP) participates in signaling pathways essential for cell homeostasis in multiple tissues, however, its function in the heart in vivo remains unknown. Here we offer the first description of the expression, function and mechanism of action of VCP in the mammalian heart in vivo in both normal and stress conditions. By using a transgenic (TG) mouse with cardiac-specific overexpression (3.5-fold) of VCP, we demonstrate that VCP is a new and powerful mediator of cardiac protection against cell death in vivo, as evidenced by a 50% reduction of infarct size after ischemia/reperfusion versus wild type. We also identify a novel role of VCP in preserving mitochondrial respiration and in preventing the opening of mitochondrial permeability transition pore in cardiac myocytes under stress. In particular, by genetic deletion of inducible isoform of nitric oxide synthase (iNOS) from VCP TG mouse and by pharmacological inhibition of iNOS in isolated cardiac myocytes, we reveal that an increase of expression and activity of iNOS in cardiomyocytes by VCP is an essential mechanistic link of VCP-mediated preservation of mitochondrial function. These data together demonstrate that VCP may represent a novel therapeutic avenue for the prevention of myocardial ischemia.

Aims: Valosin-containing protein (VCP) participates in signaling pathways essential for cell homeostasis in multiple tissues, yet its role in mammalian heart remains largely unknown. Our recent studies showed that overexpression of VCP protects cardiomyocytes from stress- induced cell death in both in vitro and in vivo , however, it is unknown which target genes and pathways are regulated by VCP through which the heart may be protected from stress-induced injury. Here we provided the first in vivo evidence of the VCP-regulated target genes by microarray gene expression profiling in a transgenic mouse model. Methods and results: We generated a transgenic (TG) mouse that over-expressed 3.5 fold VCP specifically in the heart. Total RNA extracted from the heart tissues from both adult VCP TG and their litter-matched wild type (WT) mice (N=4/group) was assayed by using Affymetrix Mouse Genome 430 2.0 Array, which interrogates over 39,000 transcripts. Differentially expressed genes (DEGs) were defined by p-value ≤ 0.05 and FC (fold change) ≥ 1.5, and totally 581 DEGs were identified between VCP TG and WT. Applying a more stringent criteria (FDR ≤ 0.05 and FC ≥ 1.5), we identified 21 upregulated and 28 downregulated genes in VCP TG mouse hearts compared to WT. Among the top DEGs, there is a significant up-regulation in the genes regulating the stress response and oxidation in VCP TG versus WT, such as a kinase interacting protein by 2.6 folds which is a key regulator of cardiac stress; glutathione peroxidase 1 by 2.2 folds which is an antioxidant enzyme and anti-apoptotic mediators: protein kinase C delta by 2.3 folds and B-cell lymphoma 2 by 2.7 folds. VCP TG mice also exhibits an upregulation in the genes involved in cellular structure and contractility, e.g., alpha 1 actin by 3.3 folds, myosin light chain 9 by 8.2 folds and Tropomyosin 2 by 4.5 folds which is a gene regulating the binding of myosin and actin. Other DEGs include the upregulated-genes regulating cellular amino acid metabolism such as pterin-4 alpha-carbinolamine dehydratase by 9.7folds, phosphatidic acid phosphatase 2c by 3.4 folds and sortilin 1 by 2.4 folds. Conclusion: Significant difference of gene regulation exists between VCP TG and WT in the heart, which may be the mechanism of VCP-mediated cardiac protection.

Background: An increase in aortic stiffness is a fundamental component of hypertension. However, the molecular mechanism involved is unclear. Our hypothesis is that the increased aortic stiffness in hypertension is partially due to the activation of serum response factor (SRF) /myocardin in vascular smooth muscle cells (VSMCs). Methods: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured by a Millar catheter and by Doppler imaging echocardiography, respectively. VSMCs were isolated from thoracic aorta (TA), then cultured and tested at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Results: Aortic pressure was higher in SHR than WKY (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, p p p p p In vitro , CCG-100602, a specific inhibitor of SRF/myocardin interaction, not only strikingly repressed the increase of the expression in SRF, myocardin and the downstream targets α-SMA and SM22, but also blocked the nuclear translocation of myocardin in TA VSMCs from SHR ( p In vivo , 2-week treatment with CCG-100602 significantly reduced MAP and ASI by 19% and 63% in SHR, respectively ( p Conclusion: The activation of myocardin/SRF in aortic VSMCs presents a novel mechanism of increased aortic stiffness in SHR.

Systemic hypertension, which eventually results in heart failure, renal failure or stroke, is a common chronic human disorder that particularly affects elders. Although many signaling pathways involved in the development of hypertension have been reported over the past decades, which has led to the implementation of a wide variety of anti-hypertensive therapies, one half of all hypertensive patients still do not have their blood pressure controlled. The frontier in understanding the molecular mechanisms underlying hypertension has now advanced to the level of epigenomics. Particularly, increasing evidence is emerging that DNA methylation and histone modifications play an important role in gene regulation and are involved in alteration of the phenotype and function of vascular cells in response to environmental stresses. This review seeks to highlight the recent advances in our knowledge of the epigenetic regulations and mechanisms of hypertension, focusing on the role of DNA methylation and histone modification in the vascular wall. A better understanding of the epigenomic regulation in the hypertensive vessel may lead to the identification of novel target molecules that, in turn, may lead to novel drug discoveries for the treatment of hypertension.

The pressure overload induced cardiac hypertrophy is a key risk factor for heart failure. However, the mechanism involved remains largely unknown. We previously showed that overexpression of valosin-containing protein (VCP) promotes cardiomyocytes survival in vitro . Here we tested the hypothesis