Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.

Ubiquitin ligase Wwp1 gene deletion attenuates diastolic dysfunction in pressure-overload hypertrophy.

Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and accompanied by abnormal extracellular matrix (ECM) accumulation. The E3 ubiquitin ligase WWP1 is a fundamental determinant ECM turnover. We tested the hypothesis that genetic ablation of Wwp1 would alter the progression of LVPO-induced HFpEF. LV echocardiography in mice with global Wwp1 deletion (n = 23; Wwp1-/-) was performed at 12 wk of age (baseline) and then at 2 and 4 wk following LVPO (transverse aortic banding) or surgery without LVPO induction. Age-matched wild-type mice (Wwp1+/+; n = 23) underwent identical protocols. LV EF remained constant and unchanged with LVPO and LV mass increased in both groups but was lower in the Wwp1-/- mice. With LVPO, the E/A ratio, an index of LV filling, was 3.97 ± 0.46 in Wwp1+/+ but was 1.73 ± 0.19 in the Wwp1-/- group (P < 0.05). At the transcriptional level, mRNA for fibrillar collagens (types I and III) decreased by approximately 50% in Wwp1-/- compared with the Wwp1+/+ group at 4 wk post-LVPO (P < 0.05) and was paralleled by a similar difference in LV fibrillar collagen content as measured by histochemistry. Moreover, mRNA levels for determinants favoring ECM accumulation, such as transforming growth factor (TGF), increased with LVPO, but were lower in the Wwp1-/- group. The absence of Wwp1 reduced the development of left ventricular hypertrophy and subsequent progression to HFpEF. Modulating the WWP1 pathway could be a therapeutic target to alter the natural history of HFpEF.NEW & NOTEWORTHY Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and is accompanied by abnormal extracellular matrix (ECM) accumulation. It is now recognized that the ECM is a dynamic entity that is regulated at multiple post-transcriptional levels, including the E3 ubiquitin ligases, such as WWP1. In the present study, WWP1 deletion in the context of an LVPO stimulus reduced functional indices of HFpEF progression and determinants of ECM remodeling.

Functional resilience of C57BL/6J mouse heart to dietary fat overload.

Molecular mechanisms underlying cardiac dysfunction and subsequent heart failure in diabetic cardiomyopathy are incompletely understood. Initially we intended to test the role of G protein-coupled receptor kinase 2 (GRK2), a potential mediator of cardiac dysfunction in diabetic cardiomyopathy, but found that control animals on HFD did not develop cardiomyopathy. Cardiac function was preserved in both wild-type and GRK2 knockout animals fed high-fat diet as indicated by preserved left ventricular ejection fraction (LVEF) although heart mass was increased. The absence of cardiac dysfunction led us to rigorously evaluate the utility of diet-induced obesity to model diabetic cardiomyopathy in mice. Using pure C57BL/6J animals and various diets formulated with different sources of fat-lard (32% saturated fat, 68% unsaturated fat) or hydrogenated coconut oil (95% saturated fat), we consistently observed left ventricular hypertrophy, preserved LVEF, and preserved contractility measured by invasive hemodynamics in animals fed high-fat diet. Gene expression patterns that characterize pathological hypertrophy were not induced, but a modest induction of various collagen isoforms and matrix metalloproteinases was observed in heart with high-fat diet feeding. PPARα-target genes that enhance lipid utilization such as Pdk4, CD36, AcadL, and Cpt1b were induced, but mitochondrial energetics was not impaired. These results suggest that although long-term fat feeding in mice induces cardiac hypertrophy and increases cardiac fatty acid metabolism, it may not be sufficient to activate pathological hypertrophic mechanisms that impair cardiac function or induce cardiac fibrosis. Thus, additional factors that are currently not understood may contribute to the cardiac abnormalities previously reported by many groups.NEW & NOTEWORTHY Dietary fat overload (DFO) is widely used to model diabetic cardiomyopathy but the utility of this model is controversial. We comprehensively characterized cardiac contractile and mitochondrial function in C57BL6/J mice fed with lard-based or saturated fat-enriched diets initiated at two ages. Despite cardiac hypertrophy, contractile and mitochondrial function is preserved, and molecular adaptations likely limit lipotoxicity. The resilience of these hearts to DFO underscores the need to develop robust alternative models of diabetic cardiomyopathy.

Overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

Qingda granule attenuates angiotensin II-induced cardiac hypertrophy and apoptosis and modulates the PI3K/AKT pathway.

Qingda granule (QDG), simplified from Qingxuan Jiangya Decoction, is a well-known traditional Chinese medicine formula that has been used for decades to treat hypertension. However, the cardioprotective effects of QDG on Ang II-induced hypertension remain unknown. This study aimed to investigate the effects of QDG on hypertension-induced cardiac hypertrophy and apoptosis, as well as explore its underlying mechanisms. Mice were infused with Ang II (500 ng/kg/min) or saline solution as control, then administered oral QDG (1.145 g/kg/day) or saline for two weeks. QDG treatment attenuated the elevation in blood pressure caused by Ang II, as well as the decreased left ventricle ejection fractions and fractional shortening. Moreover, QDG treatment significantly alleviated the Ang II-induced elevation of the ratio of heart weight to tibia length, as well as cardiac injury, hypertrophy, and apoptosis. In cultured H9C2 cells stimulated with Ang II, QDG partially reversed the increase in cell surface area and number of apoptotic cells, up-regulation of hypertrophy markers ANP and BNP, and activation of caspases-9 and -3. QDG also partially reversed Ang II-induced accumulation of reactive oxygen species (ROS), depolarization of the mitochondrial membrane, release of cytochrome C, up-regulation of Bax, and decrease in levels of p-PI3K, p-AKT, and Bcl-2. These results suggest that QDG can significantly attenuate Ang II-induced hypertension, cardiac hypertrophy and apoptosis, and it may exert these effects in part by suppressing ROS production and activating the PI3K/AKT signaling pathway.

NADPH oxidase-4 promotes eccentric cardiac hypertrophy in response to volume overload.

AIMS: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. METHODS AND RESULTS: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 µm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A. CONCLUSION: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

Xanthohumol attenuates isoprenaline-induced cardiac hypertrophy and fibrosis through regulating PTEN/AKT/mTOR pathway.

Emerging evidence suggests the cardiovascular protective effects of Xanthohumol (Xn), a prenylated flavonoid isolated from the hops (Humulus lupulus L.). However, the cardioprotective effect of Xn remains unclear. Present study aimed to investigate the protective role of Xn against isoprenaline (ISO)-induced cardiac hypertrophy and fibrosis, and elucidate the underlying mechanism. The cardiac hypertrophy and fibrosis model were established via subcutaneously administration of ISO. ISO reduced the left ventricular contractile function and elevated myocardial enzyme levels, suggesting cardiac dysfunction. Moreover, the increased cardiac myocyte area, heart weight/body weight (HW/BW) ratio and ANP/BNP expressions indicated the ISO-induced hypertrophy, while the excessive collagen-deposition and up-regulation of fibrosis marker protein (α-SMA, Collagen-I/III) expression indicated the ISO-induced fibrosis. The ISO-induced cardiac dysfunction, hypertrophy and fibrosis were significantly attenuated by oral administrated with Xn. PTEN/AKT/mTOR pathway has been reported to involve in pathogenesis of cardiac hypertrophy and fibrosis. We found that Xn administration up-regulated PTEN expression and inhibited the phosphorylation of AKT/mTOR in ISO-treated mice. Moreover, treating with VO-ohpic, a specific PTEN inhibitor, abolished the cardioprotective effect of Xn. Collectively, these results suggested that Xn attenuated ISO-induced cardiac hypertrophy and fibrosis through regulating PTEN/AKT/mTOR pathway.

The piRNA CHAPIR regulates cardiac hypertrophy by controlling METTL3-dependent N6-methyladenosine methylation of Parp10 mRNA.

PIWI-interacting RNAs (piRNAs) are abundantly expressed during cardiac hypertrophy. However, their functions and molecular mechanisms remain unknown. Here, we identified a cardiac-hypertrophy-associated piRNA (CHAPIR) that promotes pathological hypertrophy and cardiac remodelling by targeting METTL3-mediated N6-methyladenosine (m6A) methylation of Parp10 mRNA transcripts. CHAPIR deletion markedly attenuates cardiac hypertrophy and restores heart function, while administration of a CHAPIR mimic enhances the pathological hypertrophic response in pressure-overloaded mice. Mechanistically, CHAPIR-PIWIL4 complexes directly interact with METTL3 and block the m6A methylation of Parp10 mRNA transcripts, which upregulates PARP10 expression. The CHAPIR-dependent increase in PARP10 promotes the mono-ADP-ribosylation of GSK3ß and inhibits its kinase activity, which results in the accumulation of nuclear NFATC4 and the progression of pathological hypertrophy. Hence, our findings reveal that a piRNA-mediated RNA epigenetic mechanism is involved in the regulation of cardiac hypertrophy and that the CHAPIR-METTL3-PARP10-NFATC4 signalling axis could be therapeutically targeted for treating pathological hypertrophy and maladaptive cardiac remodelling.

Deletion of obscurin immunoglobulin domains Ig58/59 leads to age-dependent cardiac remodeling and arrhythmia.

Obscurin comprises a family of giant modular proteins that play key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to essential modulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban (PLN). Importantly, missense mutations localized within the obscurin-Ig58/59 region that affect binding to titins and/or PLN have been linked to the development of myopathy in humans. To elucidate the pathophysiological role of this region, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and determined the consequences of this manipulation on cardiac morphology and function under conditions of acute stress and through the physiological process of aging. Our studies show that young Obscn-ΔIg58/59 mice are susceptible to acute ß-adrenergic stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop left ventricular hypertrophy that progresses to dilation, contractile impairment, atrial enlargement, and arrhythmia as a function of aging with males being more affected than females. Experiments in ventricular cardiomyocytes revealed altered Ca2+ cycling associated with changes in the expression and/or phosphorylation levels of major Ca2+ cycling proteins, including PLN, SERCA2, and RyR2. Taken together, our work demonstrates that obscurin-Ig58/59 is an essential regulatory module in the heart and its deletion leads to age- and sex-dependent cardiac remodeling, ventricular dilation, and arrhythmia due to deregulated Ca2+ cycling.

Neuraminidase-1 promotes heart failure after ischemia/reperfusion injury by affecting cardiomyocytes and invading monocytes/macrophages.

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (ß-GAL) and protective-protein/cathepsin (PPC) A, which cleaves sialic-acids from cell surface glycoconjugates. We investigated the role of NEU1 in the myocardium after ischemia/reperfusion (I/R). Three days after inducing I/R, left ventricles (LV) of male mice (3 months-old) displayed upregulated neuraminidase activity and increased NEU1, ß-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Lin-CD11b+F4/80+Ly-6Chigh), and more anti-inflammatory macrophages (Lin-CD11b+F4/80+Ly-6Clow) 3 days after I/R, and less LV dysfunction 14 days after I/R. WT mice transplanted with hNEU1-bone marrow (BM) and hNEU1 mice with WT-BM showed significantly better LV function 14 days after I/R compared with WT mice with WT-BM. Mice with a cardiomyocyte-specific NEU1 overexpression displayed no difference in inflammation 3 days after I/R, but showed increased cardiomyocyte hypertrophy, reduced expression and mislocalization of Connexin-43 in gap junctions, and LV dysfunction despite a similar infarct scar size to WT mice 14 days after I/R. The upregulation of NEU1 after I/R contributes to heart failure by promoting inflammation in invading monocytes/macrophages, enhancing cardiomyocyte hypertrophy, and impairing gap junction function, suggesting that systemic NEU1 inhibition may reduce heart failure after I/R.

Screening for Fabry Disease in patients with unexplained left ventricular hypertrophy.

Fabry Disease (FD) is a systemic disorder that can result in cardiovascular, renal, and neurovascular disease leading to reduced life expectancy. FD should be considered in the differential of all patients with unexplained left ventricular hypertrophy (LVH). We therefore performed a prospective screening study in Edmonton and Hong Kong using Dried Blood Spot (DBS) testing on patients with undiagnosed LVH. Participants found to have unexplained LVH on echocardiography were invited to participate and subsequently subjected to DBS testing. DBS testing was used to measure α-galactosidase (α-GAL) enzyme activity and for mutation analysis of the α-galactosidase (GLA) gene, both of which are required to make a diagnosis of FD. DBS testing was performed as a screening tool on patients (n = 266) in Edmonton and Hong Kong, allowing for detection of five patients with FD (2% prevalence of FD) and one patient with hydroxychloroquine-induced phenocopy. Left ventricular mass index (LVMI) by GLA genotype showed a higher LVMI in patients with IVS4 + 919G > A mutations compared to those without the mutation. Two patients were initiated on ERT and hydroxychloroquine was discontinued in the patient with a phenocopy of FD. Overall, we detected FD in 2% of our screening cohort using DBS testing as an effective and easy to administer screening tool in patients with unexplained LVH. Utilizing DBS testing to screen for FD in patients with otherwise undiagnosed LVH is clinically important due to the availability of effective therapies and the value of cascade screening in extended families.

Preoperative myocardial expression of E3 ubiquitin ligases in aortic stenosis patients undergoing valve replacement and their association to postoperative hypertrophy.

Currently, aortic valve replacement is the only treatment capable of relieving left ventricle pressure overload in patients with severe aortic stenosis. It aims to improve cardiac function and revert hypertrophy, by triggering myocardial reverse remodeling. Despite immediately relieving afterload, reverse remodeling turns out to be extremely variable. Among other factors, the extent of reverse remodeling may depend on how well ubiquitin-proteasome system tackle hypertrophy. Therefore, we assessed tagged ubiquitin and ubiquitin ligases in the left ventricle collected from patients undergoing valve replacement and tested their association to the degree of reverse remodeling. Patients were classified according to the regression of left ventricle mass (ΔLVM) and assigned to complete (ΔLVM≥15%) or incomplete (ΔLVM≤5%) reverse remodeling groups. No direct inter-group differences were observed. Nevertheless, correlation analysis supports a fundamental role of the ubiquitin-proteasome system during reverse remodeling. Indeed, total protein ubiquitination was associated to hypertrophic indexes such as interventricular septal thickness (r = 0.55, p = 0.03) and posterior wall thickness (r = 0.65, p = 0.009). No significant correlations were observed for Muscle Ring Finger 3. Surprisingly, though, higher levels of atrogin-1 were associated to postoperative interventricular septal thickness (r = 0.71, p = 0.005). In turn, Muscle Ring Finger 1 correlated negatively with this postoperative hypertrophy marker (r = -0.68, p = 0.005), suggesting a cardioprotective role during reverse remodeling. No significant correlations were found with left ventricle mass regression, although a trend for a negative association between the ligase Murine Double Minute 2 and mass regression (r = -0.44, p = 0.10) was found. Animal studies will be necessary to understand whether this ligase is protective or detrimental. Herein, we show, for the first time, an association between the preoperative myocardial levels of ubiquitin ligases and postoperative hypertrophy, highlighting the therapeutic potential of targeting ubiquitin ligases in incomplete reverse remodeling.

The ubiquitin ligase WWP1 contributes to shifts in matrix proteolytic profiles and a myocardial aging phenotype with diastolic heart.

Ubiquitylation is a key event that regulates protein turnover, and induction of the ubiquitin ligase E3 WWP1 has been associated with age. Left ventricular hypertrophy (LVH) commonly occurs as a function of age and can cause heart failure (HF) with a preserved ejection fraction (EF; HFpEF). We hypothesized that overexpression (O/E) of WWP1 in the heart would cause LVH as well as functional and structural changes consistent with the aging HFpEF phenotype. Global WWP1 O/E was achieved in mice (n = 11) and echocardiography (40 MHz) performed to measure LV mass, EF, Doppler velocities (early E, late/atrial A), myocardial relaxation (E'), and isovolumetric relaxation time (IVRT) at 4, 6, and 8 wk. Age-matched wild-type animals (n = 15) were included as referent controls. LV EF was identical (60 ± 1 vs. 60 ± 1%, P > 0.90) with no difference in LV mass (67 ± 3 vs. 75 ± 5, P > 0.25) at 4 wk. However, at 8 wk of age, LV mass increased over twofold, E/A fell (impaired passive filling), and E/E' was lower and IVRT prolonged (impaired LV relaxation) - all P < 0.05. Collagen percent area increased by over twofold and fibrillar collagen expression (RT-PCR) over 1.5-fold (P < 0.05) with WWP1 O/E. WWP1 with an anti-WWP1 antibody could be identified in isolated cardiac fibroblasts, with WWP1 increased over twofold in O/E fibroblasts (P < 0.05). Inducing WWP1 expression caused LVH and preserved systolic function but impaired diastolic dysfunction, consistent with the HFpEF phenotype. Targeting the WWP1 pathway may be a novel therapeutic target for this intractable form of HF associated with aging.NEW & NOTEWORTHY Heart failure (HF) with a preserved ejection fraction (HFpEF) is a growing cause of HF and commonly afflicts the elderly. Milestones for HFpEF include diastolic dysfunction and an abnormal extracelluar matrix (ECM). The ubiquitin ligases, such as WWP1, change with aging and regulate critical protein turnover/stability processes, such as the ECM. The present study demonstrated that induction of WWP1 in mice induced LV hypertrophy, diastolic dysfunction, and ECM accumulation, consistent with the HFpEF phenotype, and thus may identify a new therapeutic pathway.

Histone H4R3 symmetric di-methylation by Prmt5 protects against cardiac hypertrophy via regulation of Filip1L/ß-catenin.

BACKGROUND AND PURPOSE: Although histone lysine methylation has been extensively studied for their participation in pathological cardiac hypertrophy, the potential regulatory role of histone arginine methylation remains to be elucidated. The present study focused on H4R3 symmetric di-methylation (H4R3me2s) induced by protein arginine methyltransferase 5 (Prmt5), and explored its epigenetic regulation and underlying mechanisms in cardiomyocyte hypertrophy. METHODS AND RESULTS: 1. The expressions of Prmt5 and H4R3me2s were suppressed in cardiac hypertrophy models in vivo and in vitro; 2. Prmt5 silencing or its inhibitor EPZ, or knockdown of cooperator of Prmt5 (Copr5) to disrupt H4R3me2s, facilitated cardiomyocyte hypertrophy, whereas overexpression of wild type Prmt5 rather than the inactive mutant protected cardiomyocytes against hypertrophy; 3. ChIP-sequence analysis identified Filip1L as a target gene of Prmt5-induced H4R3me2s; 4. Knockdown or inhibition of Prmt5 impaired Filip1L transcription and subsequently prevented ß-catenin degradation, thus augmenting cardiomyocyte hypertrophy. CONCLUSIONS: The present study reveals that Prmt5-induced H4R3me2s ameliorates cardiomyocyte hypertrophy by transcriptional upregulation of Filip1L and subsequent enhancement of ß-catenin degradation. Deficiency of Prmt5 and the resulting suppression of H4R3me2s might facilitate the development of pathological cardiac hypertrophy. Prmt5 might serve as a key epigenetic regulator in pathological cardiac hypertrophy.

ß-adrenergic activation may promote myosin light chain kinase degradation through calpain in pressure overload-induced cardiac hypertrophy: ß-adrenergic activation results in MLCK degradation.

BACKGROUND: ß-adrenergic activation is able to exacerbate cardiac hypertrophy. Myosin light chain kinase (MLCK) and its phosphorylated substrate, phospho-myosin light chain 2 (p-MLC2), play vital roles in regulating cardiac hypertrophy. However, it is not yet clear whether there is a relationship between ß-adrenergic activation and MLCK in the progression of cardiac hypertrophy. Therefore, we explored this relationship and the underlying mechanisms in this work. METHODS: Cardiac hypertrophy and cardiomyocyte hypertrophy were induced by pressure overload and isoproterenol (ISO) stimulation, respectively. Echocardiography, histological analysis, immunofluorescence and qRT-PCR were used to confirm the successful establishment of the models. A ß-blocker (metoprolol) and a calpain inhibitor (calpeptin) were administered to inhibit ß-adrenergic activity in rats and calpain in cardiomyocytes, respectively. The protein expression levels of MLCK, myosin light chain 2 (MLC2), p-MLC2, myosin phosphatase 2 (MYPT2), calmodulin (CaM) and calpain were measured using western blotting. A cleavage assay was performed to assess the degradation of recombinant human MLCK by recombinant human calpain. RESULTS: The ß-blocker alleviated cardiac hypertrophy and dysfunction, increased MLCK and MLC2 phosphorylation and decreased calpain expression in pressure overload-induced cardiac hypertrophy. Additionally, the calpain inhibitor calpeptin attenuated cardiomyocyte hypertrophy, upregulated MLCK and p-MLC2 and reduced MLCK degradation in ISO-induced cardiomyocyte hypertrophy. Recombinant human calpain degraded recombinant human MLCK in vitro in concentration- and time-dependent manners, and this degradation was inhibited by the calpain inhibitor calpeptin. CONCLUSION: Our study suggested that ß-adrenergic activation may promote the degradation of MLCK through calpain in pressure overload-induced cardiac hypertrophy.

Stachydrine hydrochloride suppresses phenylephrine-induced pathological cardiac hypertrophy by inhibiting the calcineurin/nuclear factor of activated T-cell signalling pathway.

The calcineurin (CaN)/nuclear factor of activated T-cell (NFAT) signalling pathway plays an important role in pathological cardiac hypertrophy. Here, we investigated the potential effects of stachydrine hydrochloride, a bioactive constituent extracted from the Chinese herb Leonurus japonicus Houtt. (Yimucao), on pathological cardiac hypertrophy during chronic α1-adrenergic receptor (α1-AR) activation and the underlying mechanisms. First, by transcriptome analysis, we determined that pathological hypertrophy models could be prepared after phenylephrine stimulation. In primary cultured neonatal rat ventricular myocytes, stachydrine hydrochloride reduced phenylephrine-induced cardiomyocyte surface area and the mRNA expression of cardiac hypertrophy biomarkers (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and ß-myosin heavy chain/α-myosin heavy chain (ß-MHC/α-MHC)). In addition, phenylephrine stimulation potently induced activation of the CaN/NFAT pathway. Interestingly, stachydrine hydrochloride inhibited CaN activation and reduced NFATc3 nuclear translocation in phenylephrine-stimulated neonatal rat ventricular myocytes. In mice treated with phenylephrine, stachydrine hydrochloride treatment decreased cardiac hypertrophy and regulated heart function. Collectively, our data show that stachydrine hydrochloride decreases cardiac hypertrophy in phenylephrine-stimulated hearts by inhibiting the CaN/NFAT pathway, which might contribute to alleviation of pathological cardiac hypertrophy and cardiac dysfunction by stachydrine hydrochloride after phenylephrine stimulation This also indicated that governing of CaN/NFAT pathway might serve as a preventive or therapeutic strategy for pathological cardiac hypertrophy.

Exogenous supplement of glucagon like peptide-1 protects the heart against aortic banding induced myocardial fibrosis and dysfunction through inhibiting mTOR/p70S6K signaling and promoting autophagy.

Mammalian target of rapamycin (mTOR) and a ribosomal protein S6 kinase (p70S6K) mediate tissue fibrosis and negatively regulate autophagy. This study aims to investigate whether glucagon-like peptide-1 (GLP-1) analog liraglutide protects the heart against aortic banding-induced cardiac fibrosis and dysfunction through inhibiting mTOR/p70S6K signaling and promoting autophagy activity. Male SD rats were randomly divided into four groups (n = 6/each group): sham operated control; abdominal aortic constriction (AAC); liraglutide treatment during AAC (0.3 mg/kg, injected subcutaneously twice daily); rapamycin treatment during AAC (0.2 mg/kg/day, administered by gastric gavage). Relative to the animals with AAC on week 16, liraglutide treatment significantly reduced heart/body weight ratio, inhibited cardiomyocyte hypertrophy, and augmented plasma GLP-1 level and tissue GLP-1 receptor expression. Phosphorylation of mTOR/p70S6K, populations of myofibroblasts and synthesis of collagen I/III in the myocardium were simultaneously inhibited. Furthermore, autophagy regulating proteins: LC3-II/LC3-I ratio and Beclin-1 were upregulated, and p62 was downregulated by liraglutide. Compared with liraglutide group, treatment with rapamycin, a specific inhibitor of mTOR, compatibly augmented GLP-1 receptor level, inhibited phosphorylation of mTOR/p70S6K and expression of p62 as well as increased level of LC3-II/LC3-I ratio and Beclin-1, suggesting that there is an interaction between GLP-1 and mTOR/p70S6K signaling in the regulation of autophagy. In line with these modifications, treatment with liraglutide and rapamycin significantly reduced perivascular/interstitial fibrosis, and preserved systolic/diastolic function. These results suggest that the inhibitory effects of liraglutide on cardiac fibrosis and dysfunction are potentially mediated by inhibiting mTOR/p70S6K signaling and enhancing autophagy activity.

Peroxiredoxin-1 ameliorates pressure overload-induced cardiac hypertrophy and fibrosis.

BACKGROUND: Previous studies have demonstrated that Peroxiredoxin 1 (Prdx1) is a modulator of physiological and pathophysiological cardiovascular events. However, the roles of Prdx1 in cardiac hypertrophy and heart failure (HF) have barely been explored. Thus, this study aimed to investigate whether Prdx1 participates in cardiac hypertrophy and to elucidate the possible associated mechanisms. METHODS: Mice were subjected to transverse aortic constriction (TAC) for four weeks to induce pathological cardiac hypertrophy. Cardiomyocyte-specific Prdx1 overexpression in mice was achieved using an adeno-associated virus system. Morphological examination; echocardiography; and hemodynamic, biochemical and histological analyses were used to evaluate the roles of Prdx1 in pressure overload-induced cardiac hypertrophy and HF. RESULTS: First, the results showed that Prdx1 expression was noticeably upregulated in hypertrophic mouse hearts and cardiomyocytes with phenylephrine (PE)-induced hypertrophy in vitro. Prdx1 overexpression exerted protective effects against cardiac hypertrophy and fibrosis and ameliorated cardiac dysfunction in mice subjected to pressure overload. In addition, Prdx1 overexpression decreased pressure overload-induced cardiac inflammation and oxidative stress. Further studies demonstrated that Prdx1 overexpression increased the levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant protein, heme oxygenase-1 (HO-1), in mice. Moreover, Nrf2 knockdown offset the antihypertrophic and anti-oxidative stress effects of Prdx1 overexpression. CONCLUSIONS: Prdx1 protects against pressure overload-induced cardiac hypertrophy and HF by activating Nrf2/HO-1 signaling. These data indicate that targeting Prdx1 may be an attractive pharmacotherapeutic strategy for the treatment of cardiac hypertrophy and HF.

Berberine Attenuates Cardiac Hypertrophy Through Inhibition of mTOR Signaling Pathway.

PURPOSE: Berberine was reported to exert beneficial effects on cardiac hypertrophy. However, its cellular and molecular mechanisms still remained unclear. METHODS: Cardiac hypertrophy was induced in male Sprague-Dawley (SD) rats by transverse aorta constriction (TAC), with or without 6-week treatment of berberine. Echocardiography was performed to evaluate cardiac function. Rats were then sacrificed for histological assay, with detection for proteins and mRNA. H9c2 cells were pretreated with berberine of different concentrations (0, 1 µM, and 10 µM), followed by treatment with 2 µM norepinephrine (NE). Cells of different groups were measured for cell surface area, with mRNA detected by qRT-PCR and proteins by western blot. RESULTS: Compared with the sham group, rats of the TAC group showed significantly increased cardiac hypertrophy and fibrosis, which could be ameliorated by treatment with berberine. Western blot showed that mammalian target of rapamycin (mTOR) signaling-related protein expressions, including phospho-mTOR, phospho-4EBP1, and phospho-p70 S6K (Thr389), but not phospho-p70 S6K (Ser371), were significantly increased in the TAC group, which were inhibited by berberine treatment. H9c2 cells were treated with NE to induce hypertrophy with increased cell surface area and mRNA expressions of anp and bnp. Berberine of 10 µM, but not 1 µM, significantly ameliorated NE-induced hypertrophy and inhibited protein expressions of mTOR signaling pathway similar to those in the rat model. CONCLUSIONS: Berberine can exert cardioprotective effects on both pressure-overloaded cardiac hypertrophy and failure in vivo and NE-induced hypertrophy in vitro. Our results suggest berberine could be a potential treatment for patients with cardiac hypertrophy and failure.

rAAV9-Mediated MEK1 Gene Expression Restores Post-conditioning Protection Against Ischemia Injury in Hypertrophic Myocardium.

PURPOSE: We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. METHODS: C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. RESULTS: rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3ß. CONCLUSION: These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.