Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes

Ribeiro, Alexandre J. S.; Schwab, Olivier; Mandegar, Mohammad A.; Ang, Yen‐Sin; Conklin, Bruce R.; Srivastava, Deepak; Pruitt, Beth L.

doi:10.1161/circresaha.116.310363

Cited by 105 publications

(129 citation statements)

References 52 publications

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“…Spontaneously beating cells were micropatterned onto 10 kPa hydrogels prepared with embedded fluorescent microbeads to monitor beating. Spinning disk confocal imaging was used to monitor the bead displacement as a function of time, and the total force of contraction for each cell was calculated using a MATLAB routine developed by Ribeiro et al (56) (Figure 7B-C). For each cell, the force per sarcomere was approximated by calculating the force per area.…”

Section: Resultsmentioning

confidence: 99%

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

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27Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a 28 major indicator for heart transplant. The disease is frequently caused by mutations of 29 sarcomeric proteins; however, it is not well understood how these molecular mutations 30 lead to alterations in cellular organization and contractility. To address this critical gap in 31 our knowledge, we studied the molecular and cellular consequences of a DCM mutation 32 in troponin-T, DK210. We determined the molecular mechanism of DK210 and used 33 computational modeling to predict that the mutation should reduce the force per 34 sarcomere. In mutant cardiomyocytes, we found that DK210 not only reduces contractility, 35 but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to 36 changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and 37 disease). These results link the molecular and cellular phenotypes and implicate 38 alterations in mechanosensing as an important factor in the development of DCM. 39Results 100 DK210 decreases calcium sensitivity in an in vitro motility assay 101We set out to decipher the molecular mechanism of the DK210 mutation in vitro. 102The molecular effects of cardiomyopathy mutations depend on the myosin isoform (7-9, 103 35-37) and therefore, we used porcine cardiac ventricular myosin (38). Porcine ventricular 104 cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) 105 is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to 106 human cardiac myosin, including the kinetics of the ATPase cycle, step size, and 107 sensitivity to load (38-41), making it an ideal myosin for biophysical studies. 108Given the role of troponin-T in thin filament regulation, we first determined whether 109 the DK210 mutation affects calcium-based regulation of myosin binding to thin filaments 110 using an in vitro motility assay (42). Reconstituted thin filaments, consisting of porcine 111 cardiac actin and recombinantly expressed human troponin and tropomyosin, were added 112 to a flow cell coated with porcine cardiac myosin in the presence of ATP. The speed of 113 filament translocation was measured as a function of added calcium. As has been 114 reported previously, the speed of regulated thin filament translocation increased 115 sigmoidally with increasing Ca 2+ concentration (43), ( Figure 1B). Data were fit with the Hill 116 equation to obtain the pCa50 (i.e., the concentration of calcium necessary for half-117 maximal activation). Consistent with previous studies using mouse cardiac, rabbit cardiac, 118 and rabbit skeletal muscle fibers (31, 33, 44), DK210 shows a right-shifted curve (pCa50 119 = 5.7 ± 0.1) compared to the WT (pCa50 = 6.1 ± 0.1; p < 0.0001), meaning more calcium 120 is needed for the same level of activation. This suggests that the mutant could show 121 impaired force production during a calcium transient. 122 7 123 Molecular mechanism of DK210-induced changes in thin filament regulat...

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Section: Resultsmentioning

confidence: 99%

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

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“…The complete methodology has been previously described (58). In brief, day >30 iPS-CMs from BAG3 KO1 lines were thawed on fibronectin-coated wells and recovered for 5 days before seeding on micropatterned polyacrylamide substrates.…”

Section: Methodsmentioning

confidence: 99%

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

Judge¹,

Pérez-Bermejo²,

Truong³

et al. 2017

JCI Insight

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“…Our findings from this study suggest that RhoA-ROCK is the primary mechanism of β AR-regulation of breast cancer cell mechanotype. β AR signaling is established to increase the contractility of cardiac myocytes (71) and to decrease the traction stresses of human airway smooth muscle cells (72). Recent findings show that activating β AR in adipose cells results in increased NMII activity and cell contractility, albeit through a Ca 2+ -MLCK pathway (73).…”

Section: Discussionmentioning

confidence: 99%

β-adrenergic signaling modulates cancer cell mechanotype through a RhoA-ROCK-myosin II axis

Kim

Vazquez-Hidalgo

Abdou

et al. 2019

Preprint

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The ability of cells to deform and generate forces are key mechanical properties that are implicated in metastasis. While various soluble and mechanical cues are known to regulate cancer cell mechanical phenotype or mechanotype, our knowledge of how cells translate external signals into changes in mechanotype is still emerging. We previously discovered that activation of β -adrenergic signaling, which results from soluble stress hormone cues, causes cancer cells to be stiffer or less deformable; this stiffer mechanotype was associated with increased cell motility and invasion. Here, we characterize how β -adrenergic activation is translated into changes in cellular mechanotype by identifying molecular mediators that regulate key components of mechanotype including cellular deformability, traction forces, and nonmuscle myosin II (NMII) activity. Using a micropillar assay and computational modelling, we determine that β AR activation increases cellular force generation by increasing the number of actin-myosin binding events; this mechanism is distinct from how cells increase force production in response to matrix stiffness, suggesting that cells regulate their mechanotype using a complementary mechanism in response to stress hormone cues. To identify the molecules that modulate cellular mechanotype with β AR activation, we use a high throughput filtration platform to screen the effects of pharmacologic and genetic perturbations on β AR regulation of whole cell deformability. Our results indicate that β AR activation decreases cancer cell deformability and increases invasion by signaling through RhoA, ROCK, and NMII. Our findings establish β AR-RhoA-ROCK-NMII as a primary signaling axis that mediates cancer cell mechanotype, which provides a foundation for future interventions to stop metastasis.

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Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes

Cited by 105 publications

References 52 publications

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

β-adrenergic signaling modulates cancer cell mechanotype through a RhoA-ROCK-myosin II axis

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