AimsIncreased 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.Methods and resultsFour 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.ConclusionsSRF/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.

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.

SummaryHypertension‐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.

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.

SummaryAortic 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.

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.

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration.

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.