Suppression Of Cardiac Hypertrophy Research Articles

A PRMT5 selective inhibitor EPZ015666 inhibits pressure overload-induced left ventricular dysfunction through the suppression of cardiac hypertrophy and fibrosis

A PRMT5 selective inhibitor EPZ015666 inhibits pressure overload-induced left ventricular dysfunction through the suppression of cardiac hypertrophy and fibrosis

Left Ventricular Longitudinal Strain as a Marker for Point of No Return in Hypertensive Heart Failure Treatment

There are currently no therapies that can improve prognosis in cases of heart failure (HF) with preserved ejection fraction (EF). We hypothesized that there is a point of no return after which no response to treatment is noted and that for the prevention of hypertensive HF this point may be determined by left ventricle (LV) strain, in the prevention of hypertensive HF. Here an angiotensin-converting enzyme inhibitor (ACE-I) was initiated based on myocardial strain imaging and its effects were determined in an animal model. Thirty-two male Dahl salt-sensitive rats, age 6weeks, were divided into six experimental groups and compared with low-salt (n=8) and high-salt control groups (n=8). In the early treatment group, ACE-I was administered from the age of 6weeks (n=4); in the longitudinal strain (LS) group, at 10-12weeks when LS impairment was >-21% (n=4); in the circumferential strain (CS) group, at 16-18weeks when CS impairment was >-18% (n=4); and in the EF group, at 20weeks when EF was <55% (n=4). Subsequently, all rats were sacrificed at 23weeks age, the LV and lung weight were measured, and pathologic analyses were performed. At 23weeks of age, the lung and LV weights increased in the high-salt control, EF, and CS groups, whereas the lung and LV weights in the LS and early groups were similar to those in the low-salt control group. The percentage of area of subendocardial fibrosis was >6% in the high-salt control, EF, and CS groups and <3% in the LS, early, and low-salt groups. Serial echocardiography demonstrated LS improvement in the LS group; however, the CS and EF groups showed no differences. Heart failure-related lung congestion was prevented when ACE-I was administered soon after LS impairment, accompanied by suppression of cardiac hypertrophy and fibrosis, thereby suggesting that the point of no return of myocardial remodeling due to hypertension was present after LS but before CS impairment.

Characterization of the Contribution of Retinoic Acid Receptor Isoforms in the Suppression of Cardiac Hypertrophy

Pathway analyses of proteomic studies in guinea pig and rat hearts subjected to pressure overload-induced hypertrophy and heart failure (HF) suggest altered retinoid signaling may contribute to HF progression. In recent studies, we showed that cardiac levels of the hormone all-trans retinoic acid (ATRA) are deficient in human non-ischemic HF patients. This has prompted us to revisit an old, but poorly understood, observation that ATRA suppresses phenylephrine (Phe)-induced hypertrophy in neonatal rat ventricular myocytes (NRVMs). Using cross-sectional area (CSA) as an index of hypertrophy, we show, consistent with prior work, that ATRA suppresses Phe-induced increases in NRVM CSA by average of 70%. We then used the pan-CYP26 inhibitor talarozole (Tala) to raise endogenous ATRA levels, and observed that Tala similarly inhibited Phe-induced increases in NRVM CSA (p<0.001). ATRA exerts its biological actions through a class of ligand-dependent transcription factors called retinoic acid receptors (RARs), of which there are three forms, α, β and γ. We examined the impact of RARα, RARβ, and RARγ antagonists on ATRA and Tala mediated hypertrophy suppression. The RARβ antagonist LE135 (1 μ M) fully blocked the action of ATRA (p<0.0001), while initial studies with the RARα and RARγ antagonists (BMS 195614 (1 μ M) and MM 11253 (1 μ M)) failed to significantly mitigate CSA suppression. Thus, initial pharmacologic studies indicate ATRA elicits an anti-hypertrophic program, at least in part, through activation of RARβ. Interestingly, when experiments were repeated using Tala, hypertrophy inhibition appeared to be mediated by a combination of RARβ and RARγ. Ongoing experiments are aimed at corroborating these observations in NRVMs virally-transduced with siRNA to each of RAR isoform. These studies will provide key insights to delineate the mechanisms by which ATRA dysregulation contributes to HF pathogenesis.

Irisin promotes cardiac progenitor cell-induced myocardial repair and functional improvement in infarcted heart.

Irisin, a newly identified hormone and cardiokine, is critical for modulating body metabolism. New evidence indicates that irisin protects the heart against myocardial ischemic injury. However, whether irisin enhances cardiac progenitor cell (CPC)-induced cardiac repair remains unknown. This study examines the effect of irisin onCPC-induced cardiac repair when these cells are introduced into the infarcted myocardium. Nkx2.5+ CPC stable cells were isolated from mouse embryonic stem cells. Nkx2.5 + CPCs (0.5 × 10 6 ) were reintroduced into the infarcted myocardium using PEGlylated fibrin delivery. The mouse myocardial infarction model was created by permanent ligation of the left anterior descending (LAD) artery. Nkx2.5 + CPCs were pretreated with irisin at a concentration of 5 ng/ml in vitro for 24 hr before transplantation. Myocardial functions were evaluated by echocardiographic measurement. Eight weeks after engraftment, Nkx2.5 + CPCs improved ventricular function as evident by an increase in ejection fraction and fractional shortening. These findings are concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Transplantation of Nkx2.5 + CPCs promoted cardiac regeneration and neovascularization, which were increased with the pretreatment of Nkx2.5 + CPCs with irisin. Furthermore, irisin treatment promoted myocyte proliferation as indicated by proliferative markers Ki67 and phosphorylated histone 3 and decreased apoptosis. Additionally, irisin resulted in a marked reduction of histone deacetylase 4 and increased p38 acetylation in cultured CPCs. These results indicate that irisin promoted Nkx2.5 + CPC-induced cardiac regeneration and functional improvement and that irisin serves as a novel therapeutic approach for stem cells in cardiac repair.

Abstract 586: Early Characterization of the Mechanisms of ATRA-mediated Suppression of Cardiac Hypertrophy

Pathway analyses of proteomic studies in guinea pig and rat hearts subjected to pressure overload-induced hypertrophy and heart failure (HF) suggest aberrant retinoid signaling may contribute to HF progression. Though the precise mechanisms are unknown, we recently showed that cardiac levels of the hormone all-trans retinoic acid (ATRA) are deficient in human non-ischemic HF patients. This has prompted us to revisit an old, but poorly understood, observation that ATRA suppresses phenylephrine (Phe)-induced hypertrophy in neonatal rat ventricular myocytes (NRVMs). Using confocal microscopy to measure cross-sectional area (CSA) as an index of hypertrophy, we show, consistent with prior work, that ATRA suppresses Phe-induced increases in NRVM CSA by average of 70%. Specifically, treatment groups of maintenance media (MM) alone and MM+Phe+ATRA were significantly different from the MM+Phe treatment group (p&lt;0.005, n&gt;58). ATRA exerts its biological actions through a class of ligand-dependent transcription factors called retinoic acid receptors (RARs), of which there are three forms, a, b and g. To better understand the mechanism by which ATRA exerts its anti-hypertrophic effect in NRVMs, we examined the impact of RARα, RARβ, and RARγ antagonists. The RARβ antagonist LE135 (1mM) fully blocked the action of ATRA (p&lt;0.0001, n&gt;46), while initial studies with the RARa and RARg antagonists (BMS 195614 and MM 11253) failed to significantly mitigate CSA suppression by ATRA. Thus, initial pharmacologic studies indicate ATRA elicits an anti-hypertrophic program, at least in part, through its activation of RARb. Ongoing experiments are aimed at corroborating these observations in NRVMs virally-transduced with shRNA to knock down levels of each of the RAR isoforms. These studies will provide key insights as we begin to delineate the mechanisms by which ATRA dysregulation contributes to HF pathogenesis.

Inhibition of Oct 3/4 mitigates the cardiac progenitor-derived myocardial repair in infarcted myocardium.

BackgroundRecent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit+ cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart.Methodsc-kit+ CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs.ResultsTwo weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit+ CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium.ConclusionOur results indicate that CSCs-derived cardiac regeneration improves the restoration of cardiac function and is mediated through Oct 3/4.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0252-5) contains supplementary material, which is available to authorized users.

MiR-21-3p regulates cardiac hypertrophic response by targeting histone deacetylase-8.

Growing evidences indicate that microRNAs (miRNAs) are involved in cardiac hypertrophy development. Multiple miRNAs have been identified as diagnostic and prognostic biomarkers of cardiac hypertrophy, as well as potential therapeutic tools. The present study aimed to investigate the functions and regulatory mechanisms of miR-21-3p in cardiac hypertrophy. Decreased expression of miR-21-3p was observed in cardiac hypertrophy induced by transverse aortic constriction (TAC) and angiotensin (Ang) II infusion in mice. To further explore the role of miR-21-3p in cardiac hypertrophy, rAAV-miR-21-3p was administered intravenously in mice. Overexpression of miR-21-3p markedly suppressed TAC-induced cardiac hypertrophy and also blocked Ang II-induced cardiac hypertrophy as determined by cardiac function measurement and biomarker detection. Furthermore, western blot assays showed that histone deacetylase-8 (HDAC8) was silenced by miR-21-3p, and luciferase reporter assays showed that miR-21-3p binds to the 3' UTR of HDAC8. Moreover, re-expression of HDAC8 attenuated miR-21-3p-mediated suppression of cardiac hypertrophy by enhancing phospho-Akt and phospho-Gsk3β expression. Our data reveal a role of miR-21-3p in regulating HDAC8 expression and Akt/Gsk3β pathway, and suggest that modulation of miR-21-3p levels may provide a therapeutic approach for cardiac hypertrophy.

Abstract 19320: Protein Kinase G Oxidation Blunts Its Suppression of Cardiac Hypertrophy by Diminishing Phosphorylation of Tuberin and Consequent Autophagy

Stimulation of protein kinase G (PKG) suppresses maladaptive cardiac hypertrophy in part by blunting signaling cascades such as calcineurin/NFAT (Cn) and calcium-calmodulin dependent kinase II (CamKII). Here we report PKG stimulation also enhances autophagy as an anti-hypertrophic mechanism, identify a novel phosphorylation target for this effect, and show this signaling is blunted when PKG becomes oxidized at cysteine 42 (C42). Mice with knock-in expression of mutant PKG (C42S) or littermate controls were subjected to pressure-overload. C42S mice were protected, with reduced hypertrophy and fibrosis, improved function, and blunted Cn, CamKII, and hypertrophic gene activation. Neonatal rat ventricular myocytes (NRVMs) expressing C42S-PKG exposed to 48hr endothelin-1 (ET1) also displayed less hypertrophic responses. In both C42S mice and NRVMs expressing C42S, autophagic flux was increased over WT, determined by autophagic flux assays, which was associated with inhibited mammalian target of rapamycin (mTOR) signaling. Control mice improved responses similar to C42S mice if autophagic flux was stimulated by inhibiting mTOR with everolimus. Analogous findings were obtained by mTOR suppression in NRVMs. To determine how PKG activation influences mTOR-regulated autophagy, we performed unbiased phospho-proteome analysis using mass spectrometry that revealed a unique PKG targeted site on tuberin (TSC2) at S1343. To test the functional importance of this site, we expressed a mutated TSC2, serine to glutamic acid (phospho-mimetic, S1343E), which depressed mTOR signaling, increased autophagic flux, and blunted hypertrophic signaling and cell enlargement stimulated by ET1 in NRVMs. By contrast, the cardio-protection elicited by PKG C42S was lost when TSC2 or a downstream critical autophagy protein, ATG5 were silenced. Taken together, these results provide the first evidence that PKG activation augments autophagy via targeting of TSC2 as a mechanism of anti-hypertrophic signaling, and that this occurs much more when PKG is in a reduced state (e.g. no C42-disulfide). Loss of this mechanism in myocardium under oxidative stress may play a role in the decline in protection from PKG in cardiac disease which often involves an oxidative environment.

Heme Arginate Suppresses Cardiac Lesions and Hypertrophy in Deoxycorticosterone Acetate-Salt Hypertension

In hypertension, elevated levels of oxidative/inflammatory mediators including nuclear factor kappaB (NF-kappaB), activating protein (AP-1), c-Jun-NH2-terminal kinase (JNK), and cell-regulatory proteins such as transforming growth factor beta (TGF-beta), trigger the mobilization of extracellular matrix (ECM) leading to fibrosis, hypertrophy and impairment of cardiac function. Although the heme oxygenase (HO) system is cytoprotective, its effects on cardiac fibrosis and hypertrophy in deoxycorticosterone acetate (DOCA-salt) hypertension are not completely elucidated. Here, we report cardioprotection by the HO inducer, heme arginate against histopathological lesions in DOCA-hypertension. Treatment with heme arginate restored physiological blood pressure, and abated cardiac hypertrophy (3.75 +/- 0.12 vs. 3.19 +/- 0.09 g/kg body wt; n =16, P < 0.01), left-to-right ventricular ratio (6.67 +/- 0.62 vs. 4.39 +/- 0.63; n = 16, P < 0.01), left ventricular mass (2.48 +/- 0.14 vs. 2.01 +/- 0.09 g/kg body wt; n = 16, P < 0.01) and left-ventricular wall thickness (2.82 +/- 0.16 vs. 1.98 +/- 0.14 mm; n = 16, P < 0.01), whereas the HO inhibitor, chromium mesoporphyrin, exacerbated hypertrophy and cardiac lesions. The suppression of cardiac hypertrophy was accompanied by a robust increase in HO-1, HO activity, cyclic guanosine monophosphate (cGMP), ferritin and the total antioxidant capacity, whereas 8-isoprostane, NF-kappaB, JNK, AP-1, TGF-beta, fibronectin and collagen-I were significantly abated. Correspondingly, histopathological parameters that depict progressive cardiac damage, including fibrosis, interstitial/perivascular collagen deposition, scarring, muscle-fiber thickness, muscular hypertrophy and coronary-arteriolar thickening were abated. Our study suggests that upregulating the HO system lowers blood pressure, potentiates the antioxidant status in tissues, suppresses oxidative stress/mediators such as NF-kappaB, AP-1 and cJNK, and suppresses the mobilization of ECM proteins like TGF-beta, collagen and fibronectin, with corresponding reduction of cardiac histopathological lesion and hypertrophy.

Rescuing Cardiac Malfunction

See related articles, pages 1473–1482 In response to various hypertrophic stimuli, the heart undergoes alterations in structure and function. One mechanism by which the heart adapts to physiological and pathological stimuli is an increase in size of individual cardiac myocytes, and when those stimuli are removed, the size of the myocytes can decrease. Modulation of cardiac size thus requires a very careful balance between protein synthesis and protein degradation, and any imbalance in these systems could be of potential damage to the heart. Increased protein synthesis in hypertrophied hearts is accompanied by “protein quality control” to eliminate aggregated unfolded proteins, but a failure of this monitoring system eventually leads to cardiac dysfunction.1 Heat shock proteins (HSPs) have chaperone-like properties that can bind to unfolded proteins and prevent their denaturation and aggregation.2 In recent genome surveys of mice, rats, and humans, small-molecular-weight heat shock proteins (sHSPs ≈15 to 30 kDa) have been identified. αB crystalline (CryAB) is a member of the sHSPs that is expressed at high levels in cardiomyocytes.2 CryAB has chaperone-like properties and also binds to both desmin and cytoplasmic actin which helps to maintain cytoskeletal integrity. Previous studies have shown that a mutation in CryAB mimics a form of desmin-related cardiomyopathy.3 Overexpressing CryAB in cultured cardiomyocytes and in transgenic mouse hearts protects against ischemia/reperfusion–induced cell death4,5; on the other hand, double knockout of the small heat shock proteins CryAB/SHSP2 induces abnormal cardiac growth and defective myocardial relaxation.6 In this issue of Circulation Research , Kumarapeli et al7 address and confirm the roles of CryAB in response to …

Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins

Cardiac hypertrophy is a major cause of human morbidity and mortality. Although much is known about the pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. Atrogin-1, also known as muscle atrophy F-box, is an F-box protein that inhibits pathologic cardiac hypertrophy by participating in a ubiquitin ligase complex that triggers degradation of calcineurin, a factor involved in promotion of pathologic hypertrophy. Here we demonstrated that atrogin-1 also disrupted Akt-dependent pathways responsible for physiologic cardiac hypertrophy. Our results indicate that atrogin-1 does not affect the activity of Akt itself, but serves as a coactivator for members of the Forkhead family of transcription factors that function downstream of Akt. This coactivator function of atrogin-1 was dependent on its ubiquitin ligase activity and the deposition of polyubiquitin chains on lysine 63 of Foxo1 and Foxo3a. Transgenic mice expressing atrogin-1 in the heart displayed increased Foxo1 ubiquitylation and upregulation of known Forkhead target genes concomitant with suppression of cardiac hypertrophy, while mice lacking atrogin-1 displayed the opposite physiologic phenotype. These experiments define a role for lysine 63-linked ubiquitin chains in transcriptional coactivation and demonstrate that atrogin-1 uses this mechanism to disrupt physiologic cardiac hypertrophic signaling through its effects on Forkhead transcription factors.

Abstract 1068: Genetic Evidence for a Central Role of cGMP-Dependent Protein Kinase I Alpha in Regulating Cardiac Hypertrophy In Vivo

The nitric oxide-cGMP-dependent protein kinase pathway is a central regulator of cardiovascular physiology. Protein Kinase G I (PKGI), a principle mediator of this pathway, has been implicated as a negative regulator of cardiac hypertrophy, but the specific mechanisms involved are unknown. To test the hypothesis that PKGIα regulates cardiac hypertrophy, we characterized the cardiac phenotype of mice homozygous for a mutant form of PKGIα in which critical amino acids in the N-terminal leucine zipper (LZ) motif have been substituted to disrupt PKGIα LZ binding to specific downstream effector proteins. In the unstressed state, male PKG Iα leucine zipper mutant ( LZM) mice develop progressive LV hypertrophy compared with wild type (WT) littermates with LV mass/tibia length 12.3% greater at 30 weeks of age (p=0.05), and 27% greater at 60 weeks (p=0.001). Compared with WT littermates, the hearts of 30 week old PKGIα mutants are hypercontractile with decreased end systolic diameter (p=0.02) and increased fractional shortening on echocardiography (p=0.03). Invasive hemodynamics demonstrate that LZM mice also have increased LV systolic pressure (p=0.04), developed pressure (p=0.05), and LV dP/dt max (p=0.13). To evaluate the response to hemodynamic stress, cardiac hypertrophy was induced by transaortic constriction (TAC) in male WT and LZM mice. TAC resulted in early mortality in the LZM mice (60%) compared to the WT (19%) mice at 21 days post procedure (p=0.008), with evidence of accelerated LV hypertrophy in the mice that died early (LV mass/tibia length 9.8 mg/mm in the early LZM deaths vs 7.2 mg/mm in WT survivors, p&lt;0.001). Additionally, the LZM early deaths had evidence of congestive heart failure with increased RV mass (RV mass/ tibia length 1.8 mg/mm in LZM early deaths vs 1.2 mg/mm in WT survivors, p&lt;0.05), and increased lung mass (23.1 mg/mm LZM early deaths vs 12.5 mg/mm in WT survivors, p&lt;0.005). These findings support that the N-terminal LZ domain of PKGIα is required for suppression of cardiac hypertrophy. The early mortality following TAC in the LZM mice also suggests a critical role for PKGIα in attenuating pathologic cardiac remodeling, identifying PKGIα as an attractive candidate drug target for prevention of cardiac hypertrophy and failure.

A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.