(Table 3) Stable isotope record of Cretaceous foraminifera from DSDP Hole 44-392A

Mid-Cretaceous (Barremian-Turonian) plankton preserved in deep-sea marl, organic-rich shale, and pelagic carbonate hold an important record of how the marine biosphere responded to short- and long-term changes in the ocean-climate system. Oceanic anoxic events (OAEs) were short-lived episodes of organic carbon burial that are distinguished by their widespread distribution as discrete beds of black shale and/or pronounced carbon isotopic excursions. OAE1a in the early Aptian (~120.5 Ma) and OAE2 at the Cenomanian/Turonian boundary (~93.5 Ma) were global in their distribution and associated with heightened marine productivity. OAE1b spans the Aptian/Albian boundary (~113-109 Ma) and represents a protracted interval of dysoxia with multiple discrete black shales across parts of Tethys (including Mexico), while OAE1d developed across eastern and western Tethys and in other locales during the latest Albian (~99.5 Ma). Mineralized plankton experienced accelerated rates of speciation and extinction at or near the major Cretaceous OAEs, and strontium isotopic evidence suggests a possible link to times of rapid oceanic plateau formation and/or increased rates of ridge crest volcanism. Elevated levels of trace metals in OAE1a and OAE2 strata suggest that marine productivity may have been facilitated by increased availability of dissolved iron. The association of plankton turnover and carbon isotopic excursions with each of the major OAEs, despite the variable geographic distribution of black shale accumulation, points to widespread changes in the ocean-climate system. Ocean crust production and hydrothermal activity increased in the late Aptian. Faster spreading rates [and/or increased ridge length] drove a long-term (Albian-early Turonian) rise in sea level and CO2-induced global warming. Changes in ocean circulation, water column stratification, and nutrient partitioning lead to a reorganization of plankton community structure and widespread carbonate (chalk) deposition during the Late Cretaceous. We conclude that there were important linkages between submarine volcanism, plankton evolution, and the cycling of carbon through the marine biosphere

Abstract 129: Human Mesenchymal Stem Cells Augment Contractile Function of Human Engineered Cardiac Tissues

Mesenchymal stem cells (MSC) have demonstrated efficacy for improving cardiomyocyte (CM) function in vitro, in vivo and in clinical trials, but the mechanism of this enhancement remains elusive. The objective of this study was to test the hypothesis that human engineered cardiac tissues (hECT) offer a viable model system to investigate the effects of human MSC on CM contractile function. Human CM (hCM) were produced from embryonic stem cells (hESC, H7 line) using a small-molecule based differentiation approach. Blebbistatin and BMP4 were added to hESC suspended in StemPro34 differentiation media for 24 h, followed by BMP4 and Activin A to day 4.5, followed by addition of IWR-1 Wnt inhibitor for at least 4 days. To create hECT, approximately 1 million hCM were mixed with 2.0 mg/ml bovine type I collagen and 0.9 mg/ml Matrigel, and pipetted into a mold fabricated from polydimethylsiloxane with integrated cantilever end-posts. To model hMSC cell therapy, two types of hECT were created: hCM-only control hECT, and hMSC-CM hybrid hECT containing hCM mixed with 5-10% of human bone marrow-derived MSC. Over several days in culture, the hECT self-assembled and started beating; end-post deflection was tracked in real time to compute twitch force using beam theory. Human CMs were produced with high efficiency (>70% cTnT+) with a predominantly ventricular phenotype (MLC2v+). Resulting hECTs exhibited spontaneous beating (1.3±0.4 Hz), cellular alignment, registered sarcomeres, and expression of cardiac specific genes cTnT, α-MHC, β-MHC and SERCA2a. After 11±2 days in culture, developed stress (force/area) was over 10-fold higher in hMSC-CM hybrid tissues (0.27±0.048 mN/mm 2 ) compared to hCM-only controls (0.02±0.006 mN/mm 2 ; p=0.04, n=5 per group). This reflected significantly greater twitch force (0.11±0.004 mN vs 0.033±0.016 mN, p=0.016) and smaller cross-sectional area (0.19±0.12 mm 2 vs 0.49±0.10 mm 2 ; p=0.003) in hMSC-CM hybrid vs hCM-only hECT. In conclusion, human ECT offer a novel system to study MSC-CM interactions. The findings suggest hMSC supplementation improves contractility compared to CM-only hECT. Investigating the mechanisms of hMSC-mediated enhancement of hECT function may yield insights into MSC-based therapies for cardiac regeneration

Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy

<div><p>Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS), which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT) model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP) gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new <i>in vitro</i> model for studying intrinsic mechanisms and screening new therapeutic approaches for this lethal form of heart disease.</p></div

Twitch force characteristics of wild-type and BRAF-mutant hECTs.

<p>(<b>A</b>) Cartoon of iPSC-derived hECT force data illustrating twitch characteristic parameters. (<b>B-G</b>) Mean (±SD) twitch parameters for wild-type (open bars, n = 7) and BRAF mutant (solid bars, n = 5) iPSC-hECTs tested on culture days 6 and 11, including developed force (<b>B</b>), 50% twitch duration (<b>C</b>), time to 50% contraction (<b>D</b>), time to 50% relaxation (<b>E</b>) and the maximum rates of contraction (<b>F</b>) and relaxation (<b>G</b>). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Electrical properties of of wild-type and BRAF-mutant hECTs.

<p>(<b>A</b>) Developed force versus frequency relationship of iPSC-derived hECTs at culture day 6 (left) and day 11 (right); (<b>B</b>) maximum pacing frequency of wild-type and mutant tissues; (<b>C</b>) spontaneous beating frequency; (<b>D</b>) The minimum voltage necessary for pacing for both tissues; (<b>E</b>) Relative chronotropic response to isoproterenol (normalized to baseline beating frequency) with fitted nonlinear regression model. (<b>F</b>) Twitch rate variability plots of each tissue type with a paced control for comparison. Each color represents a different tissue. (<b>G</b>) Quantification of twitch rate variability by the mean distance of each point on the twitch rate variability plots from each cluster centroid. Error bars represent standard error (<b>A</b>) or standard deviation (<b>B-G</b>) Open symbols and bars are wild-type hECTS (n = 7), filled symbols and bars are BRAF-mutant hECTs (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Characterization of wild-type and BRAF iPSC-hECTs.

<p>(<b>A</b>) Sorting paradigm for construction of defined iPSC-hECTs; (<b>B</b>) Photograph of wild-type (left) and mutant (right) tissues after 6 days in culture; (<b>C</b>) longitudinal section of wild-type (left) and mutant (right) tissues stained with hematoxylin and eosin after 12 days in culture; (<b>D</b>) Post deflections of both tissue types on day 6 at 2 Hz pacing frequency; (<b>E</b>) Cross-sectional area (mean±SD) of wild-type (open bars, n = 7) and mutant (filled bars, n = 4) tissues; (<b>F</b>) Molecular analysis of mutant and wild-type tissues performed on day 12 (n = 3 for each tissue type).</p

Investigating longitudinal changes in hECT functional phenotype.

<p>(<b>A</b>) Principal component analysis and k-means clustering of tissues paced at 2 Hz on day 6 (left) and day 11 (right). (<b>B</b>) Developed force of the wild-type tissues increased significantly over day 6 and day 11, while mutant tissues did not. The lack of a significant change from day 6 to day 11 appeared to be due to a greater increase in diastolic force of mutants from day 6 to 11 than wild-type tissues, while systolic force increased approximately by the same amount for both tissue types. (<b>C</b>) Passive Young’s modulus determined by uniaxial stretch measurements on days 12–15 (n = 4 per tissue type). * p < 0.05 between tissues types, † p < 0.05 between day 6 and day 11 for wild-type tissues.</p

Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity

Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility