Hypertrophic Phenotype Research Articles

BMP7 reduces the fibrocartilage chondrocyte phenotype through MMP2

Purpose: The fibrocartilage chondrocyte phenotype has been recognized as a pathological chondrocyte in osteoarthritic (OA) articular cartilage. Although relatively rare and mainly found in late-stage OA, the fibrocartilage chondrocyte expresses a high ratio of genes related to unfavorable OA outcomes, emphasizing its importance in OA disease development. Development of the fibrocartilage chondrocyte phenotype resembles fibrotic processes also observed in other tissues. BMP7 is being explored as a potential disease-modifying molecule reversing the hypertrophic chondrocyte phenotype in OA.

Circ-Zfp644 acts as a pro-hypertrophic mediator in an Ang-II induced in vitro myocardial hypertrophy model.

Cardiac hypertrophy (CH) is a common risk factor for heart failure and even sudden cardiac death. Molecules have emerged as vital regulators in cardiac disorders. LIM domain kinase 1 (Limk1) is reported as a pro-fibrotic mediator in patients with permanent atrial fibrillation and it has also been suggested to aggravate cardiac dysfunction in rats with chronic heart failure. The present study observed that Limk1 was significantly upregulated in the in vitro model of CH induced by angiotensin II (Ang-II). Interestingly, silencing Limk1 led to inhibition ofthe hypertrophic phenotypes in Ang-II-treated cardiomyocytes. Next, through a series of mechanistic assays including RIP assay, RNA pull-down assay, and luciferase reporter assay, miR-93-5p was verified to target Limk1. Furthermore, circ-Zfp644 was validated as the sponge of miR-93-5p. Circ-Zfp644 functioned as a ceRNA to upregulate Limk1 expression via sequestering miR-93-5p in Ang-II-treated cardiomyocytes. Finally, a range of rescue assays revealed that circ-Zfp644 stimulated hypertrophic effects in Ang-II-treated cardiomyocytes via upregulating Limk1 while miR-93-5p exerted the opposite effects via its inhibition on Limk1. On the whole, the present study revealed that circ-Zfp644 aggravated CH through modulating the miR-93-5p/Limk1 axis. The findings observed on the in vitro model of CH provided new potential for developing CH treatment.

Torpor versus interbout arousal: what is more important for protein metabolism and regeneration?

Hibernation is used by specie as diverse as ground squirrels, snakes and bats to cope with reduced food availability and lowered temperatures and is a unique model for studying the molecular mechanisms of plasticity in striated muscles. Important questions in modern muscle biology are why some skeletal muscles atrophy and other muscles such as the diaphragm and the heart hypertrophy during hibernation, and why, despite the atrophy, skeletal muscle strength and fatigue resistance are maintained after arousal. The molecular mechanisms responsible are of interest both from a fundamental biological viewpoint and as a potential therapy for patients in critical care units. In this issue of Experimental Physiology, Yan et al. (2021) shed light on the distinctive features of the molecular mechanisms involved in the development of the hypertrophic response in the diaphragm and the atrophic response in the gastrocnemius muscle in hibernating Daurian ground squirrels. In particular, the authors showed that a decrease in protein synthesis, an increase in protein degradation and a decrease in muscle regeneration potential contributed to the atrophy of the gastrocnemius muscles during torpor, while an increase in protein synthesis, a decrease in protein degradation and an increase in muscle regeneration potential contributed to diaphragm muscle hypertrophy. In discussing these new and interesting results, it can be assumed, however, that a significant contribution to the formation of hypertrophic or atrophic muscle phenotype could have been made by changes in anabolic and catabolic processes occurring in interbout arousals, as indicated by the results of recent studies (Chang et al., 2020; Popova et al., 2020 ). Further research is needed to show how important interbout arousals are in the regulation of protein turnover and regeneration in different striated muscles in hibernating animals. Understanding these and other molecular mechanisms is important for choosing strategies and methods for treatment of skeletal muscle pathologies and diseases of the cardiovascular system, as well as for avoiding injurious interventions in cases of energy deficits in critical illness (Stanzani et al., 2020). None declared.

Whole-Exome Sequencing and hiPSC Cardiomyocyte Models Identify MYRIP, TRAPPC11, and SLC27A6 of Potential Importance to Left Ventricular Hypertrophy in an African Ancestry Population.

Background: Indices of left ventricular (LV) structure and geometry represent useful intermediate phenotypes related to LV hypertrophy (LVH), a predictor of cardiovascular (CV) disease (CVD) outcomes.Methods and Results: We conducted an exome-wide association study of LV mass (LVM) adjusted to height2.7, LV internal diastolic dimension (LVIDD), and relative wall thickness (RWT) among 1,364 participants of African ancestry (AAs) in the Hypertension Genetic Epidemiology Network (HyperGEN). Both single-variant and gene-based sequence kernel association tests were performed to examine whether common and rare coding variants contribute to variation in echocardiographic traits in AAs. We then used a data-driven procedure to prioritize and select genes for functional validation using a human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) model. Three genes [myosin VIIA and Rab interacting protein (MYRIP), trafficking protein particle complex 11 (TRAPPC11), and solute carrier family 27 member 6 (SLC27A6)] were prioritized based on statistical significance, variant functional annotations, gene expression in the hiPSC-CM model, and prior biological evidence and were subsequently knocked down in the hiPSC-CM model. Expression profiling of hypertrophic gene markers in the knockdowns suggested a decrease in hypertrophic expression profiles. MYRIP knockdowns showed a significant decrease in atrial natriuretic factor (NPPA) and brain natriuretic peptide (NPPB) expression. Knockdowns of the heart long chain fatty acid (FA) transporter SLC27A6 resulted in downregulated caveolin 3 (CAV3) expression, which has been linked to hypertrophic phenotypes in animal models. Finally, TRAPPC11 knockdown was linked to deficient calcium handling.Conclusions: The three genes are biologically plausible candidates that provide new insight to hypertrophic pathways.

Performance of Synthetic Extracellular Volume Fraction in Different Cardiac Phenotypes From a Prospective Cohort of Patients Referred for Cardiac Magnetic Resonance.

A synthetic myocardial extracellular volume fraction (sECV) can be obtained without blood hematocrit (Hct) by using the linear relationship between Hct and the longitudinal relaxation time of blood. Concerns have been raised about the widespread clinical application of this approach. To assess the relationship between measured ECV (m-ECV) and sECV, using both a published model and a locally derived one. Single-center, prospective. A 1.5 T/modified Look Locker (MOLLI) sequence. Fifty-two healthy subjects and 113 patients (76 with and 37 without a hypertrophic cardiac phenotype). Three ECV values were obtained for each patient: 1) measured ECV (m-ECV), using Hct from a venous blood sample; 2) Fent-synthetic ECV (F-sECV), using the equation proposed by Fent et al; and 3) Local-synthetic ECV (L-sECV), using the equation obtained from a local derivation cohort comprising 83 subjects. Shapiro-Wilk test, analysis of variance, Kruskal Wallis test, Pearson correlation, Bland-Altman analysis, univariate and multivariable regression analysis. In the validation cohort (N=82), Bland-Altmann analysis revealed an excellent agreement between m-ECV and L-sECV with a statistically insignificant bias (-0.1%, limits of agreement: -2.8% and 2.6%; P=0.528), while in the overall population (N=165), the mean bias between m-ECV and F-sECV was small but significant (1.2%, limits of agreement: -1.5% and 3.9%, P < 0.05). F-sECV bias was significantly higher for measured Hct (m-Hct) values <0.372 (2.3% vs. 1.0%, P < 0.05). Among the phenotype subgroups, amyloidotic patients showed a higher bias compared to others, both with F-sECV and L-sECV (2.3% vs. 1.1%, P < 0.05 and 1.1% vs. 0.2%, P < 0.05, respectively). Although synthetic ECV performs well in an external cohort, the use of a local formula further improves the accuracy of ECV estimate over a broad spectrum of cardiac phenotypes. Local sECV performs better for a wider range of Hct values, compared to the published model. Amyloidosis is the only group associated with a lower accuracy. 5 TECHNICAL EFFICACY: Stage 2.

Individualized interactomes for network-based precision medicine in hypertrophic cardiomyopathy with implications for other clinical pathophenotypes

Progress in precision medicine is limited by insufficient knowledge of transcriptomic or proteomic features in involved tissues that define pathobiological differences between patients. Here, myectomy tissue from patients with obstructive hypertrophic cardiomyopathy and heart failure is analyzed using RNA-Seq, and the results are used to develop individualized protein-protein interaction networks. From this approach, hypertrophic cardiomyopathy is distinguished from dilated cardiomyopathy based on the protein-protein interaction network pattern. Within the hypertrophic cardiomyopathy cohort, the patient-specific networks are variable in complexity, and enriched for 30 endophenotypes. The cardiac Janus kinase 2-Signal Transducer and Activator of Transcription 3-collagen 4A2 (JAK2-STAT3-COL4A2) expression profile informed by the networks was able to discriminate two hypertrophic cardiomyopathy patients with extreme fibrosis phenotypes. Patient-specific network features also associate with other important hypertrophic cardiomyopathy clinical phenotypes. These proof-of-concept findings introduce personalized protein-protein interaction networks (reticulotypes) for characterizing patient-specific pathobiology, thereby offering a direct strategy for advancing precision medicine.

Left ventricular force adaptation and cardiac deformation in the progression of aortic stenosis

Abstract Funding Acknowledgements Type of funding sources: None. Introduction. Aortic stenosis (AS) is one of the most common valvular heart diseases; however, the association between left ventricular (LV) myocardial deformation and hemodynamic forces (HDFs) is still mostly unexplored. Purpose. This study aimed to assess the differences in LV myocardial deformation and HDFs in a large cohort of patients with aortic stenosis retrospectively. Methods. Two-hundred fifty-four subjects (median age 77 years, 50% women) with preserved LV ejection fraction (LVEF), and mild (n = 87), moderate (n = 92) or severe (n = 75) AS, were included in the study. The 2D LV global longitudinal strain (GLS), circumferential strain (GCS), and HDFs were measured with new software that allowed us to calculate all these values and parameters from the three apical views. Results. When comparing severe AS to mild AS, LV mass appeared increased while the LV hypertrophy phenotype was concentric (p &amp;lt;0.0001). Along with the progression of the AS, LVEF was decreased. All GLS, GCS, and HDFs parameters were uniformly reduced in severe AS compared to mild AS (p &amp;lt;0.0001), in the same way, LV longitudinal force, LV longitudinal systolic force, and LV impulse have proven to be accurate on ROC curves (AUC 70%, 73% and 73% respectively). Conclusion. The integrated approach of deformation and cardiac mechanics allows the description of pathophysiological changes during the progression of mild to severe aortic stenosis. Abstract Figure. Strain parameters and aortic stenosis

Small Vessel Disease: Another Component of the Hypertrophic Cardiomyopathy Phenotype Not Necessarily Associated with Fibrosis.

Background: Hypertrophic cardiomyopathy (HCM) is characterized by myocardial disarray, small vessel disease (SVD), and fibrosis. The relationship between SVD and replacement-type fibrosis is still unclear. Methods: Histopathologic assessment of replacement-type fibrosis and SVD in HCM patients with either end-stage heart failure (HF) or sudden cardiac death (SCD). Chronic ischemic heart disease (IHD) patients served as controls. Results: Forty HCM hearts, 10 HF and 30 SCD, were studied. Replacement-type fibrosis was detected in all HF and in 57% of SCD cases. In SCD, replacement-type fibrosis was associated with older age, greater septal thickness, SVD prevalence, and score (all p < 0.05). Prevalence of SVD did not show significant differences among SCD, HF, and IHD (73%, 100% and 95%, respectively), while SVD score was higher in HF than IHD and SCD (2.4, 1.95, and 1.18, respectively) and in areas with replacement-type fibrosis vs. those without in HF (3.4 vs. 1.4) and SCD (1.4 vs. 0.8) (all p < 0.05). Conclusions: SVD is a frequent feature in HCM independent of the clinical presentation. A higher SVD score is observed in HCM-HF and in areas with replacement-type fibrosis. Although SVD is part of the HCM phenotype, further remodeling of the microcirculation might occur secondarily to fibrosis.

MicroRNA-21 regulates right ventricular remodeling secondary to pulmonary arterial pressure overload

Right ventricular (RV) function is a critical determinant of survival in patients with pulmonary arterial hypertension (PAH). While miR-21 is known to associate with vascular remodeling in small animal models of PAH, its role in RV remodeling in large animal models has not been characterized. Herein, we investigated the role of miR-21 in RV dysfunction using a sheep model of PAH secondary to pulmonary arterial constriction (PAC). RV structural and functional remodeling were examined using ultrasound imaging. Our results showed that post PAC, RV strain significantly decreased at the basal region compared with t the control. Moreover, such dysfunction was accompanied by increases in miR-21 levels. To determine the role of miR-21 in RV remodeling secondary to PAC, we investigated the molecular alteration secondary to phenylephrine induced hypertrophy and miR21 overexpression in vitro using neonatal rat ventricular myocytes (NRVMs). We found that overexpression of miR-21 in the setting of hypertrophic stimulation augmented only the expression of proteins critical for mitosis but not cytokinesis. Strikingly, this molecular alteration was associated with an eccentric cellular hypertrophic phenotype similar to what we observed in vivo PAC animal model in sheep. Importantly, this hypertrophic change was diminished upon suppressing miR-21 in NRVMs. Collectively, our in vitro and in vivo data demonstrate that miR-21 is a critical contributor in the development of RV dysfunction and could represent a novel therapeutic target for PAH associated RV dysfunction.

Cardiac Amyloidosis Therapy: A Systematic Review

Heart involvement in Cardiac Amyloidosis (CA) results in a worsening of the prognosis in almost all patients with both light-chain (AL) and transthyretin amyloidosis (ATTR). The mainstream CA is a restrictive cardiomyopathy with hypertrophic phenotype at cardiac imaging that clinically leads to heart failure with preserved ejection fraction (HFpEF). An early diagnosis is essential to reduce cardiac damage and to improve the prognosis. Many therapies are available, but most of them have late benefits to cardiac function; for this reason, novel therapies are going to come soon.

Pharmacological Protein Kinase C Modulators Reveal a Pro-hypertrophic Role for Novel Protein Kinase C Isoforms in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro. Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs. Conclusion: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer’s disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.

Identifying Adverse Remodeling in Hypertrophic Cardiomyopathy Phenotypes Role of Left Atrial Parameters

Identifying Adverse Remodeling in Hypertrophic Cardiomyopathy Phenotypes Role of Left Atrial Parameters

Cardiac magnetic resonance in the assessment of hypertrophic cardiomyopathy phenotypes and stages – pictorial review

The aim of this paper is to present recent advances in hypertrophic cardiomyopathy (HCM) diagnosis and treatment based on a literature review. Special emphasis has been placed on the role of cardiac magnetic resonance imaging (CMR) for the assessment of morphological and functional consequences of different stages of HCM including prognostication. The text is illustrated with the images and data of the HCM patients diagnosed with CMR study in our hospital. CMR is an important tool, particularly relevant in novel risk factors and LV dysfunction groups. The HCM group with overt left ventricular dysfunction is underrecognized, often labelled by clinicians as dilated cardiomyopathy. Advanced diagnostic and management strategies effectively influence the natural history of HCM.

Dual-specificity Phosphatase 9 protects against Cardiac Hypertrophy by targeting ASK1.

The functions of dual-specificity phosphatase 9 (DUSP9) in hepatic steatosis and metabolic disturbance during nonalcoholic fatty liver disease were discussed in our prior study. However, its roles in the pathophysiology of pressure overload-induced cardiac hypertrophy remain to be illustrated. This study attempted to uncover the potential contributions and underpinning mechanisms of DUSP9 in cardiac hypertrophy. Utilizing the gain-and-loss-of-functional approaches of DUSP9 the cardiac phenotypes arising from the pathological, echocardiographic, and molecular analysis were quantified. The results showed increased levels of DUSP9 in hypertrophic mice heart and angiotensin II treated cardiomyocytes. In accordance with the results of cellular hypertrophy in response to angiotensin II, cardiac hypertrophy exaggeration, fibrosis, and malfunction triggered by pressure overload was evident in the case of cardiac-specific conditional knockout of DUSP9. In contrast, transgenic mice hearts with DUSP9 overexpression portrayed restoration of the hypertrophic phenotypes. Further explorations of molecular mechanisms indicated the direct interaction of DUSP9 with ASK1, which further repressed p38 and JNK signaling pathways. Moreover, blocking ASK1 with ASK1-specific inhibitor compensated the pro-hypertrophic effects induced by DUSP9 deficiency in cardiomyocytes. The main findings of this study suggest the potential of DUSP9 in alleviating cardiac hypertrophy at least partially by repressing ASK1, thereby looks promising as a prospective target against cardiac hypertrophy.

The effect of cardiac geometry variation according to sex and race on outcomes in patients with acute coronary syndrome undergoing percutaneous coronary intervention.

IntroductionThe prevalence and long-term consequences of differences in baseline cardiac geometry (as a result of hypertension) in patients undergoing percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) are ill-defined. The primary purpose of this study was to clarify whether there were differences among sexual and racial groups in echocardiographic findings reflecting cardiac geometry and adaptation in patients undergoing PCI for ACS and whether this could explain the differences in outcomes seen between these groups.Material and methodsWe analyzed 1-year follow-up data from a single institution, a retrospective, observational study that enrolled 1,153 patients who presented with ACS and were treated with PCI, for whom echocardiographic data were available.ResultsNormal, concentric hypertrophy, and eccentric hypertrophy in males vs. females were observed as follows: 29% vs. 19% (p = 0.001), 25% vs. 31% (p = 0.02), and 8% vs. 14% (p = 0.004), respectively. The primary endpoint of all-cause death (n = 89, 7.7%) occurred in 48 (10.5%) females and in 41 (8.2%) males, p = 0.03. Major adverse cardiac events and bleeding (MACE-B – all-cause death, non-fatal myocardial infarction, stroke or hospitalization for bleeding) was higher among women than men (21.6% vs. 13.5%, p = 0.0002). Males with eccentric hypertrophy (EH) had similar MACE-B outcomes as females with EH 1-year post-PCI (29% vs. 32%, respectively, p = 0.77).ConclusionsFemales undergoing PCI for ACS are at higher risk for worse outcomes because they are more likely to express the eccentric hypertrophy phenotype; however, it did not account for the difference in adverse outcomes observed between sexes.

Tenosynovial complications identify TTR cardiac amyloidosis among patients with hypertrophic cardiomyopathy phenotype.

To investigate the prevalence of CTS and BBTR in a consecutive cohort of ATTR-CA patients, compared with patients with HCM or AFD and with individuals without cardiac disease history. Consecutive patients with a diagnosis of ATTR-CA, HCM and AFD were evaluated. A control group of consecutive patients was recruited among subjects hospitalized for noncardiac reasons and no cardiac disease history. The presence of BBTR, CTS or prior surgery related to these conditions was ascertained. 342 patients were prospectively enrolled, including 168 ATTR-CA (141 ATTRwt, 27 ATTRm), 81 with HCM/AFD (N=72 and 9, respectively) and 93 controls. CTS was present in 75% ATTR-CA patients, compared with 13% and 10% of HCM/AFD and controls (P=0.0001 for both comparisons). Bilateral CTS was present in 60% of ATTR-CA patients, while it was rare (2%) in the other groups. BBTR was present in 44% of ATTR-CA patients, 8% of controls and 1% in HCM/AFD. CTS and BBTR are fivefold more prevalent in ATTR-CA patients compared with cardiac patients with other hypertrophic phenotypes. Positive predictive accuracy for ATTR-CA is highest when involvement is bilateral. Upper limb assessment of patients with HCM phenotypes is a simple and effective way to raise suspicion of ATTR-CA.

The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression

We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1β and IL-34 were investigated by RT-qPCR and ELISA. Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1β induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.

MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1

BackgroundHuman mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets.MethodsThe miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients.ResultsWithin the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found.ConclusionsAlthough miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

Mir15a/mir16-1 cluster and its novel targeting molecules negatively regulate cardiac hypertrophy.

In response to pathological stimuli, the heart develops ventricular hypertrophy that progressively decompensates and leads to heart failure. miRNAs are increasingly recognized as pathogenic factors, clinically relevant biomarkers, and potential therapeutic targets. We identified that mir15a/mir16‐1 cluster was negatively correlated with hypertrophic severity in patients with hypertrophic cardiomyopathy. The mir15a/mir16‐1 expression was enriched in cardiomyocytes (CMs), decreased in hypertrophic human hearts, and decreased in mouse hearts after transverse aortic constriction (TAC). CM‐specific mir15a/mir16‐1 knockout promoted cardiac hypertrophy and dysfunction after TAC. CCAAT/enhancer binding protein (C/EBP)β was responsible for the downregulation of mir15a/mir16‐1 cluster transcription. Mechanistically, mir15a/mir16‐1 cluster attenuated the insulin/IGF1 signal transduction cascade by inhibiting multiple targets, including INSR, IGF‐1R, AKT3, and serum/glucocorticoid regulated kinase 1 (SGK1). Pro‐hypertrophic response induced by mir15a/mir16‐1 inhibition was abolished by knockdown of insulin receptor (INSR), insulin like growth factor 1 receptor (IGF1R), AKT3, or SGK1. In vivo systemic delivery of mir15a/mir16‐1 by nanoparticles inhibited the hypertrophic phenotype induced by TAC. Importantly, decreased serum mir15a/mir16‐1 levels predicted the occurrence of left ventricular hypertrophy in a cohort of patients with hypertension. Therefore, mir15a/mir16‐1 cluster is a promising therapeutic target and biomarker for cardiac hypertrophy.

Abstract 16928: Discordant Mechanisms in Heart Failure and Hypertrophy

Introduction: Patients with heart failure and a preserved ejection fraction (HFpEF) present heart function abnormalities that remain poorly understood. Defining proteomic signature of HF that is independent of left ventricular hypertrophy (LVH) should allow for stratification of its subtypes and potential mechanism that contributes to the disease. Hypothesis: We hypothesized that HFpEF proteomic signature would be comprised of the hypertrophy and contractile protein phenotype. Methods: Intraoperative left ventricular (LV) myocardial biopsies were obtained from patients (n=21) recruited to undergo coronary artery bypass grafting (CABG). Patients were categorized to: control non-hypertensive (n=9), LVH (n=5), and HFpEF (n=7). Myocardial tissue was subfractionated: cytoplasmic- (neutral pH), myofilament- (acidic pH), and membrane-enriched extract (SDS-soluble). All fractions were assessed for protein quantity and Lys/Arg modifications using liquid chromatography mass spectrometry (LC-MS). Results: In HFpEF, 13% of the cardiac LV proteome changed compared to control heart, with a substantial proportion (77%) decreasing in quantity across all three cardiac fractions, while with LVH, 61% of the proteomic LV changes were increased. Although glycolysis and gluconeogenesis increased in both cardiopathies with respect to control, in HFpEF more subtly than in LVH. Modified proteome of the HFpEF was dominated by decreases in protein succinylation (e.g. ATP5L, THIM, IDHP, APOB, GSH1, KNTC1) and to a lesser degree in methylation (ROA3, HSP7C) or acetylation compared to control. This general trend of down-regulation of succinylation can be attributed to depletion in the levels of succinyl-CoA, the cofactor of enzymatic Lys succinylation. Importantly, there was a striking discordant activation/inhibition of cell death and proliferation pathways between the HFpEF and LVH. Two major upstream regulator clusters linked the proteome changes in cell growth and proliferation to RICTOR and Myc that showed completely opposite trends in LVH and HFpEF groups. Conclusions: HFpEF has a unique proteome signature compared to LV hypertrophy profile which does not arise from sub-proteome involved in contraction but rather is involved in overall cell death.