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5000+ ORGANOIDS
5000+ ORGANOIDS
Positive inotropic drug validation in SmartHeart® cardiac rings
Data generated with the SmartHeart assay (4Dcell Heart 3D platform) under low extracellular Ca2+ Tyrode solution
Recording conditions: Day 21 post-seeding • Low extracellular Ca2+ Tyrode solution • Spontaneous beating • Contractility extracted from brightfield videos
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HIGHLIGHTS
→ Multiparametric contractility readout (frequency, contraction strain, max contraction speed, max relaxation speed) enables compound-specific fingerprints.
→ Ring-shaped 3D architecture supports physiologically relevant force distribution and coordinated tissue-level contraction, improving interpretability versus 2D monolayers.
→ Recording at day 21 post-seeding targets a more functionally mature window where integrated beta-adrenergic and Ca2+-handling phenotypes are more likely to emerge.
→ Low [Ca2+] Tyrode conditions place tissues in a submaximal calcium state, expanding dynamic range for detecting positive inotropy.
→ Isoprenaline shows both positive chronotropy and inotropy in SmartHeart, yielding a positive force-frequency relationship that is often weak or absent in typical 2D hiPSC-CM assays.
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SUMMARY
This document provides web-page-ready content (PDF format) organized similarly to 4Dcell literature review/white paper pages, but focused specifically on positive inotropic pharmacology measured with the SmartHeart® contractility assay. Dose-response data were acquired from ring-shaped human cardiac tissues at day 21 after cell seeding and recorded in a low-calcium Tyrode solution. Contractility was quantified from brightfield videos using ring tracking to extract beating frequency (chronotropy), contraction strain and contraction speed (inotropy proxies), and relaxation speed (lusitropy proxy). The following compounds are covered: isoprenaline (isoproterenol), Bay K 8644, omecamtiv mecarbil, and istaroxime.
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INTRODUCTION
Contractility is an integrated phenotype emerging from intracellular Ca2+ cycling, myofilament activation, and the mechanical boundary conditions experienced by the tissue. As a result, the same compound can appear different depending on whether it is evaluated in a 2D monolayer (minimal load, limited 3D structure) or in an engineered tissue that develops coordinated mechanics.
The SmartHeart platform generates self-assembled ring-shaped cardiac tissues in multiwell plates. The closed-ring geometry promotes circumferential alignment and distributes forces around the ring, providing a stable and trackable deformation pattern for contractility analysis.
Because the present dataset was recorded at day 21, the measured responses reflect an engineered tissue that has undergone several weeks of self-organization and functional development. This timing is particularly relevant for beta-adrenergic and Ca2+-handling phenotypes that tend to strengthen with maturation.
Why the ring-shaped 3D structure matters
The ring geometry creates a closed, continuous tissue that contracts against its own structure, producing a consistent deformation mode that can be robustly tracked over time.
Circumferential organization and multi-cellular coupling promote coordinated contraction and relaxation, reducing edge effects that can complicate interpretation in 2D cultures.
Because the tissue experiences an internal mechanical load, changes in Ca2+ handling or myofilament kinetics are more likely to translate into measurable strain and contraction/relaxation speeds.
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METHODOLOGY
Tissue format: ring-shaped engineered human cardiac tissues (hiPSC-derived cardiomyocytes co-cultured with cardiac fibroblasts) generated using SmartHeart plates.
Readout: brightfield video-based tracking of ring deformation. Metrics reported as fold-change versus vehicle (DMSO) include beating frequency, contraction strain, max contraction speed, and max relaxation speed.
Recording conditions: day 21 after cell seeding; low extracellular Ca2+ Tyrode solution; spontaneous beating.
Why low Ca2+ Tyrode? Lowering extracellular Ca2+ reduces vehicle (DMSO) twitch amplitude and Ca2+ transient magnitude, creating a submaximal state. In this regime, positive inotropes can recruit additional contractile reserve, improving sensitivity and helping separate mechanism-specific profiles (Ca2+ entry vs myofilament activation vs Ca2+ reuptake).
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RESULTS
Table 1 summarizes the direction of effects observed in SmartHeart rings for each compound (fold-change vs vehicle (DMSO)).
Isoprenaline (Isoproterenol) - beta-adrenergic agonist
Data summary (N = 2 independent experiments; n = 16 cardiac rings): Isoprenaline produces a clear, dose-dependent increase in beating frequency. Contraction strain also increases with dose, and both max contraction and max relaxation speeds rise strongly.
Interpretation in SmartHeart: The increase in contractile metrics occurs in parallel with the increase in spontaneous rate, yielding an apparent positive force-frequency coupling across the dose range. This is consistent with an integrated beta-adrenergic response where both Ca2+ cycling and relaxation kinetics are enhanced.
Contrast with typical 2D behavior: In many 2D hiPSC-cardiomyocyte monolayer assays, beta-adrenergic agonists reliably increase beating rate but can show limited or variable increases in contraction amplitude, partly due to immature Ca2+ handling and negative force-frequency behavior. In SmartHeart rings recorded here (day 21, low Ca2+ Tyrode), rate and force-related metrics increase together, revealing beta-adrenergic inotropy and a positive force-frequency relationship.
Figure 1. Isoprenaline dose-response in SmartHeart rings (day 21, low Ca2+ Tyrode). Fold-change vs vehicle (DMSO) for beating frequency, contraction strain, max contraction speed, and max relaxation speed. N = 2 independent experiments; n = 16 cardiac rings.
Bay K 8644 - L-type Ca2+ channel agonist
Data summary (N = 1 independent experiment; n = 11 cardiac rings): Bay K 8644 decreases beating frequency across the tested concentration range, while increasing contraction strain and max contraction speed. Relaxation speed remains close to vehicle (DMSO) with a small upward trend at the highest dose.
Interpretation in SmartHeart: Bay K 8644 is expected to enhance L-type Ca2+ channel activity, increasing Ca2+ entry and supporting a positive inotropic effect. Here, the reduction in spontaneous rate occurs alongside increased mechanical output, providing a useful example where inotropy is uncoupled from chronotropy under low extracellular Ca2+ conditions. To try to explain this, we can hypothetize that at 0.6 mM [Ca²⁺], even modest increases in calcium influx via Bay K might trigger calcium-dependent inactivation of pacemaker mechanisms or activate calcium-sensitive potassium channels, slowing automaticity
Figure 2. Bay K 8644 dose-response in SmartHeart rings (day 21, low Ca2+ Tyrode). Fold-change vs vehicle (DMSO) for beating frequency, contraction strain, max contraction speed, and max relaxation speed. N = 1 independent experiment; n = 11 cardiac rings.
Omecamtiv mecarbil - cardiac myosin activator
Data summary (N = 1 independent experiment; n = 10 cardiac rings): Omecamtiv mecarbil shows minimal change in beating frequency overall (slight decrease at the highest concentration). Contraction strain increases substantially at mid-to-high concentrations. Max contraction speed increases modestly then drops below vehicle (DMSO) at the highest concentration, while relaxation speed shows a modest increase at the highest concentration.
Interpretation in SmartHeart: Omecamtiv mecarbil is a myosin activator; a typical functional signature is increased systolic performance without necessarily increasing shortening velocity. The SmartHeart data are consistent with this concept: strain increases while max contraction speed does not monotonically increase and can decrease at higher exposure, suggesting altered contraction kinetics rather than a purely Ca2+-driven acceleration.
Figure 3. Omecamtiv mecarbil dose-response in SmartHeart rings (day 21, low Ca2+ Tyrode). Fold-change vs vehicle (DMSO) for beating frequency, contraction strain, max contraction speed, and max relaxation speed. N = 1 independent experiment; n = 10 cardiac rings.
Istaroxime - luso-inotropic agent
Data summary (N = 1 independent experiment; n = 10 cardiac rings): Istaroxime produces the strongest contractility phenotype in this dataset. Beating frequency remains near vehicle (DMSO) (minor changes), while contraction strain increases markedly. Both max contraction speed and max relaxation speed rise strongly, with relaxation speed showing a pronounced dose-dependent increase.
Interpretation in SmartHeart: The combined increase in inotropy and relaxation kinetics is a classic luso-inotropic profile. In the context of low extracellular Ca2+, the strong increase in relaxation speed is particularly informative because it indicates improved calcium cycling efficiency and/or accelerate cross-bridge detachment dynamics at the tissue level.
Figure 4. Istaroxime dose-response in SmartHeart rings (day 21, low Ca2+ Tyrode). Fold-change vs vehicle (DMSO) for beating frequency, contraction strain, max contraction speed, and max relaxation speed. N = 1 independent experiment; n = 10 cardiac rings
Istaroxime - luso-inotropic agent
Data summary (N = 1 independent experiment; n = 10 cardiac rings): Istaroxime produces the strongest contractility phenotype in this dataset. Beating frequency remains near vehicle (DMSO) (minor changes), while contraction strain increases markedly. Both max contraction speed and max relaxation speed rise strongly, with relaxation speed showing a pronounced dose-dependent increase.
Interpretation in SmartHeart: The combined increase in inotropy and relaxation kinetics is a classic luso-inotropic profile. In the context of low extracellular Ca2+, the strong increase in relaxation speed is particularly informative because it indicates improved ability to clear Ca2+ and/or accelerate cross-bridge detachment dynamics at the tissue level.
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SMARTHEART VS 2D ASSAYS - PRACTICAL INTERPRETATION
Why SmartHeart can differ from 2D: Contractility readouts in 2D monolayers are typically derived from cell motion under minimal mechanical load. This can lead to strong chronotropic responses but weaker, variable, or even inverted rate-force coupling (negative force-frequency) when spontaneous rate changes. Additionally, 2D cultures often exhibit immature calcium handling and limited sarcomeric organization, which further compromises their ability to recapitulate adult-like contractile responses.
In contrast, engineered 3D tissues provide coordinated mechanics, structural alignment and more physiological boundary conditions. In this dataset, SmartHeart rings show clear positive inotropy for multiple mechanisms (beta-adrenergic stimulation, L-type Ca2+ channel agonism, myosin activation, and luso-inotropy), and isoprenaline displays an integrated rate + force phenotype consistent with a positive force-frequency relationship, a hallmark of cardiac maturity that is typically absent in 2D cultures.
Practical consequence: Simultaneous measurement of frequency, strain, and contraction/relaxation kinetics helps disambiguate whether an apparent change in "force" is driven by true inotropy, by rate shifts, or by altered twitch kinetics (e.g., myosin activators).
This multi-parametric approach provides mechanistic insight that single-endpoint assays cannot achieve.
Force-frequency relationship under beta-adrenergic stimulation
In the SmartHeart dataset, isoprenaline increases spontaneous beating frequency and, simultaneously, increases contraction strain and contraction/relaxation speeds. This joint increase produces an apparent positive force-frequency relationship across the dose range.
This observation is important because, in many 2D systems, a rate increase can dominate the phenotype and obscure true inotropy. In SmartHeart rings, integrated mechanics and the chosen recording window/conditions allow both chronotropy and inotropy to be resolved in parallel.
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WHY MEASURE CONTRACTILITY - DISEASE & TOXICOLOGY RELEVANCE