Dataset Information

Interplay of Genotype and Substrate Stiffness in Driving the Hypertrophic Cardiomyopathy Phenotype in iPSC-Micro-Heart Muscle Arrays.

ABSTRACT:

Introduction

In clinical and animal studies, Hypertrophic Cardiomyopathy (HCM) shares many similarities with non-inherited cardiac hypertrophy induced by pressure overload (hypertension). This suggests a potential role for mechanical stress in priming tissues with mutation-induced changes in the sarcomere to develop phenotypes associated with HCM, including hypercontractility and aberrant calcium handling. Here, we tested the hypothesis that heterozygous loss of function of Myosin Binding Protein C (MYBCP3 ^+/- , mutations in which account for almost 50% of inherited HCM) combines with environmental stiffness to drive HCM phenotypes.

Methods

We differentiated isogenic control (WTC) and MYBPC3 ^+/- iPSC into cardiomyocytes using small molecule manipulation of Wnt signaling, and then purified them using lactate media. The purified cardiomyocytes were seeded into "dog bone" shaped stencil molds to form micro-heart muscle arrays (μHM). To mimic changes in myocardial stiffness stemming from pressure overload, we varied the rigidity of the substrates μHM contract against. Stiffness levels ranged from those corresponding to fetal (5 kPa), healthy (15 kPa), pre-fibrotic (30 kPa) to fibrotic (65 kPa) myocardium. Substrates were embedded with a thin layer of fluorescent beads to track contractile force, and parent iPSC were engineered to express the genetic calcium indicator, GCaMP6f. High speed video microscopy and image analysis were used to quantify calcium handling and contractility of μHM.

Results

Substrate rigidity triggered physiological adaptation for both genotypes. However, MYBPC3 ^+/- μHM showed a lower tolerance to substrate stiffness with the peak traction on 15 kPa, while WTC μHM had peak traction on 30 kPa. MYBPC3 ^+/- μHM exhibited hypercontractility, which was exaggerated by substrate rigidity. MYBPC3 ^+/- μHM hypercontractility was associated with longer rise times for calcium uptake and force development, along with higher overall Ca²⁺ intake.

Conclusion

We found MYBPC3 ^+/- mutations cause iPSC-μHM to exhibit hypercontractility, and also a lower tolerance for mechanical stiffness. Understanding how genetics work in combination with mechanical stiffness to trigger and/or exacerbate pathophysiology may lead to more effective therapies for HCM.

Supplementary information

The online version contains supplementary material available at (10.1007/s12195-021-00684-x).

SUBMITTER: Guo J

PROVIDER: S-EPMC8548480 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Interplay of Genotype and Substrate Stiffness in Driving the Hypertrophic Cardiomyopathy Phenotype in iPSC-Micro-Heart Muscle Arrays.

Guo Jingxuan J Jiang Huanzhu H Oguntuyo Kasoorelope K Rios Brandon B Boodram Zoë Z Huebsch Nathaniel N

Cellular and molecular bioengineering 20210625 5

<h4>Introduction</h4>In clinical and animal studies, Hypertrophic Cardiomyopathy (HCM) shares many similarities with non-inherited cardiac hypertrophy induced by pressure overload (hypertension). This suggests a potential role for mechanical stress in priming tissues with mutation-induced changes in the sarcomere to develop phenotypes associated with HCM, including hypercontractility and aberrant calcium handling. Here, we tested the hypothesis that heterozygous loss of function of Myosin Bindin ...[more]

PMID: 34777601

Similar Datasets

Project description:Introduction: Patients with hypertrophic cardiomyopathy (HCM) are at risk for lethal ventricular arrhythmia, but the electrophysiological substrate behind this is not well-understood. We used non-invasive electrocardiographic imaging to characterize patients with HCM, including cardiac arrest survivors. Methods: HCM patients surviving ventricular fibrillation or hemodynamically unstable ventricular tachycardia (n = 17) were compared to HCM patients without a personal history of potentially lethal arrhythmia (n = 20) and a pooled control group with structurally normal hearts. Subjects underwent exercise testing by non-invasive electrocardiographic imaging to estimate epicardial electrophysiology. Results: Visual inspection of reconstructed epicardial HCM maps revealed isolated patches of late activation time (AT), prolonged activation-recovery intervals (ARIs), as well as reversal of apico-basal trends in T-wave inversion and ARI compared to controls (p < 0.005 for all). AT and ARI were compared between groups. The pooled HCM group had longer mean AT (60.1 ms vs. 52.2 ms, p < 0.001), activation dispersion (55.2 ms vs. 48.6 ms, p = 0.026), and mean ARI (227 ms vs. 217 ms, p = 0.016) than structurally normal heart controls. HCM ventricular arrhythmia survivors could be differentiated from HCM patients without a personal history of life-threatening arrhythmia by longer mean AT (63.2 ms vs. 57.4 ms, p = 0.007), steeper activation gradients (0.45 ms/mm vs. 0.36 ms/mm, p = 0.011), and longer mean ARI (234.0 ms vs. 221.4 ms, p = 0.026). A logistic regression model including whole heart mean activation time and activation recovery interval could identify ventricular arrhythmia survivors from the HCM cohort, producing a C statistic of 0.76 (95% confidence interval 0.72-0.81), with an optimal sensitivity of 78.6% and a specificity of 79.8%. Discussion: The HCM epicardial electrotype is characterized by delayed, dispersed conduction and prolonged, dispersed activation-recovery intervals. Combination of electrophysiologic measures with logistic regression can improve differentiation over single variables. Future studies could test such models prospectively for risk stratification of sudden death due to HCM.

Project description:The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (Fpassive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte Fpassive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. Fpassive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. Fpassive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered Fpassive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher Fpassive compared to control hearts and significantly lower Fpassive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced Fpassive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.

Project description:BackgroundAberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways.MethodsWe analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8).ResultsGene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor.ConclusionsHuman HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

Project description:BackgroundGenetic testing can determine family screening strategies and has prognostic and diagnostic value in hypertrophic cardiomyopathy (HCM). However, it can also pose a significant psychosocial burden. Conventional scoring systems offer modest ability to predict genotype positivity. The aim of our study was to develop a novel prediction model for genotype positivity in patients with HCM by applying machine learning (ML) algorithms.MethodsWe constructed 3 ML models using readily available clinical and cardiac imaging data of 102 patients from Columbia University with HCM who had undergone genetic testing (the training set). We validated model performance on 76 patients with HCM from Massachusetts General Hospital (the test set). Within the test set, we compared the area under the receiver operating characteristic curves (AUROCs) for the ML models against the AUROCs generated by the Toronto HCM Genotype Score (the Toronto score) and Mayo HCM Genotype Predictor (the Mayo score) using the Delong test and net reclassification improvement.ResultsOverall, 63 of the 178 patients (35%) were genotype positive. The random forest ML model developed in the training set demonstrated an AUROC of 0.92 (95% CI, 0.85-0.99) in predicting genotype positivity in the test set, significantly outperforming the Toronto score (AUROC, 0.77 [95% CI, 0.65-0.90], P=0.004, net reclassification improvement: P<0.001) and the Mayo score (AUROC, 0.79 [95% CI, 0.67-0.92], P=0.01, net reclassification improvement: P=0.001). The gradient boosted decision tree ML model also achieved significant net reclassification improvement over the Toronto score (P<0.001) and the Mayo score (P=0.03), with an AUROC of 0.87 (95% CI, 0.75-0.99). Compared with the Toronto and Mayo scores, all 3 ML models had higher sensitivity, positive predictive value, and negative predictive value.ConclusionsOur ML models demonstrated a superior ability to predict genotype positivity in patients with HCM compared with conventional scoring systems in an external validation test set.

Dataset Information

Interplay of Genotype and Substrate Stiffness in Driving the Hypertrophic Cardiomyopathy Phenotype in iPSC-Micro-Heart Muscle Arrays.

Introduction

Methods

Results

Conclusion

Supplementary information

Publications

Interplay of Genotype and Substrate Stiffness in Driving the Hypertrophic Cardiomyopathy Phenotype in iPSC-Micro-Heart Muscle Arrays.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets