To obtain beating cardiomyocytes from stem cells, we used either the hESC line H7 or skin fibroblast-derived iPSCs. Cardiomyocytes were successfully derived using a well-established method to differentiate these pluripotent stem cells to the cardiac lineage 
. These pluripotent stem cell-derived cardiomyocytes expressed the cardiac markers cardiac troponin T (cTnT), sarcomeric α-actinin, and myosin light chain 2a (MLC2a) (Fig. S1
), though their spatial organization is more rounded than rectangular as is seen in CMs obtained from heart tissue. Moreover, they beat spontaneously in vitro
(iPSC-CM in Video S1
and hESC-CM in Video S2
). To measure the force generated by single CMs, we started by calibrating the spring constant of the AFM cantilever using the thermal noise method 
. The typical spring constant for these cantilevers was around 0.04 N/m. The cantilever was brought into gentle contact with the surface of a CM until the cantilever registered a deflection corresponding to 100 pN of force (), measuring indentation (). Thereafter, we turned off feedback to the z
-piezo and measured beats (). Both iPSC- and hESC-derived cardiomyocytes contract rhythmically in the axial direction, and we noticed the force, duration and frequency vary across independent single cells (). These stem-cell-derived CMs were grown on gelatin-coated, glass-bottom petri dishes, and were firmly attached – we never observed detachment of the cells due to the AFM cantilever. We found that the iPSC-derived cardiomyocytes (iPSC-CM) beat comparably to hESC-derived cardiomyocytes (hESC-CM), with contraction forces of 0.49±0.45 nN (n
9) and 0.23±0.11 nN (n
9), respectively (p
0.29) (). The total force output of these cells may be higher than we measured, because there may be lateral modes of the contraction that are not measured by this method. These measurements were done at the single point of each cell that presented the greatest beat force; we assessed the variation of beat forces at multiple points across single cells later. The mean beat rate of iPSC-CM was 0.80±0.17 beats/s (n
9), slightly slower than that of hESC-CM at 1.06±0.23 beats/s (n
0.015). The mean beat durations were 0.26±0.06 s (n
9) and 0.19±0.05 s (n
9) for iPSC-CMs and hESC-CMs, respectively (p
0.075). Our measurements show that the CMs derived from iPSC and hESC contract with the similar mechanical properties and support the use of stem cell-derived cardiomyocytes as a model system.
Single iPSC and hESC cardiomyocytes.
During culture, the iPSC-CMs can form large clusters comprising dozens of cells () that can be measured by AFM, as shown in the beat trajectory () and in the histogram of contraction force (). The beating force of a single cell within the cluster was 2.37±0.16 nN (n
106 beats). This force was stronger than the force of solitary single cells by an order of magnitude, at least partly due to the combined movement of all the cells in the cluster. The beating force of aggregated iPSC-CMs is regular (force CV
4.8%), in contrast to isolated iPSC CMs (CV
23%). Additionally, the aggregate contracts with uniform rhythm: 1.72±0.03 beats/s (rate CV
1.7%) as compared to solitary iPSC-CM (CV
20%). The consistency of contraction force and frequency shows that CMs behave more synchronously when in contact with other CMs than when solitary. This result is consistent with the known existence of cardiac gap junctions, which allow for the spread of action potentials across CMs. Together, these results show that AFM can be used to measure solitary CM and the more physiologically relevant aggregates of CM.
AFM measurement of iPSC-CM cluster.
To demonstrate the capability of AFM to measure the effect of drugs on CMs, we used norepinephrine (NE, 4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol), a demethylated form of epinephrine that non-specifically activates both alpha-1 and beta-1 adreneregic receptors. NE has long-established effects as both a positive inotrope, and to lesser extent, a positive chronotrope as well. To study the effect of NE, we treated both solitary iPSC-CMs and hESC-CMs with NE at 100 µmol/L concentration and measured beats before treatment and immediately following treatment. As shown in the trajectories () and statistical analyses (), the contraction force of iPSC-CMs increased significantly from 0.18±0.06 nN to 0.48±0.23 nN (p<0.001) upon treatment with NE. The drug also affected the rhythm, though the chronotropic effect was weaker than the inotropic effect. After applying NE, 21% of beats were faster than a cutoff of 1.7 beats/s as compared to 6% prior to treatment (). For the hESC-CM, the contraction force increased from 0.097±0.019 nN to 0.31±0.03 nN (p<0.001) after treatment with NE, but there was minimal effect on the beat rate.
Measurement of drug effect on CMs.
We next wanted to assess whether AFM could detect the more subtle effect of dose changes of an inotrope on iPSC-CM. We treated small clusters of hESC-CM with epinephrine doses from 10 nmol/L to 32 µmol/L and measured beat forces as before. We found that beat force increased as the dose increased, with an estimated EC50 of 260 nmol/L (). This value is in agreement with previous reports 
. On average, we found a 2.6-fold increase in force comparing beats prior to treatment with epinephrine and treatment with 32 µmol/L epinephrine. To test whether our AFM method could detect inhibition of adrenergic stimulation, we pre-treated hESC-CM with metoprolol (100 nmol/L) for 1 hour, then treated the CMs with epinephrine at sequentially increasing doses from 10 nmol/L to 32 µmol/L. We saw a slight decrease of beating force upon the first treatment with epinephrine (10 nmol/L dose), perhaps due to subtle movement of the tip because of fluidic shifts when the drug was introduced. Such movements were not seen with subsequent injections of epinephrine. Importantly, no increases in beat force was observed upon treatment of epinephrine in the metoprolol-treated CMs (p
0.17 comparing doses of 10 nM to 32 µM epinephrine) (). Together these results show that AFM can be used to measure both the inotropic and chronotropic effects of drugs and inhibitors on CMs.
Because the orientation of actin-myosin filaments within a cardiomyocyte is anisotropic, different parts of the CM show different amounts of movement and contractile forces with each beat. To measure the spatial heterogeneity of contraction force, we developed a method called “dwell mapping.” By superimposing a grid on the cell, we comprehensively map the cell for elasticity by nano-indenting at each point on the grid, and for beat properties by dwelling the cantilever at each point for an interval that enables the measurement of a few beats. In practice, we sampled grids comprising 100–1000 points, most of which fell onto the cell and some of which fell onto the glass surface (). Because elasticity and height at the contact point could change during the beat cycle, these changes could lead to heterogeneity in the measured forces. Dwell mapping measures the local height and local elasticity (Young's modulus) of the cell simultaneously with the local contraction forces ().
AFM dwell map of dilated cardiomyopathy iPSC-CM.
Defects in the mechanical properties of CMs may lead to cardiomyopathies 
. Dilated cardiomyopathy (DCM) is a life-threatening genetic disorder arising from mutations of many proteins including cardiac troponin T (cTnT) 
. Cardiac troponin T binds Ca2+
, plays a critical role in the contraction of CMs, and has been shown to be critical for heart development 
. We showed in another work that iPSC-CMs derived from patients with DCM showed significantly decreased beat forces, but comparable rates and beat durations as iPSC-CMs derived from healthy siblings. The patient showed a typical clinical presentation of DCM 
. We measured dwell maps of an iPSC-CM derived from a patient with DCM and found phenotypic differences compared to a healthy iPSC-CM. The contraction force histogram and Young's modulus histogram obtained from dwell maps of the DCM iPSC-CM show bimodal distributions (, sides, DCM in blue). By contrast, the force histogram obtained from dwell maps of the healthy control iPSC-CM (, red) shows a single population of points in terms of beating force and Young's modulus. Nonparametric bootstrap analysis of all beating points on the dwell maps gives a mean force of 1.35 nM for the control cell (95% confidence interval 1.18 nN–1.54 nN) and 0.55 nN for the DCM cell (95% CI 0.48 nN–0.64 nN)(Mann-Whitney p
). Similarly, nonparametric bootstrap analysis of log-transformed Young's Moduli showed a mean elasticity of 296 Pa for the control cell (95% CI 244–367 Pa) and 167 Pa for the DCM cell (95% CI 116–235 Pa) (Mann-Whitney p
0.0006). To compare the points of the two dwell maps, we used a two-dimensional (i.e., Force, Young's Modulus) Kolmogorov-Smirnov test and found the DCM and control maps are statistically distinct (p
). These results from dwell-mapping studies show that iPSC-CM from patients with DCM show increased populations of points of low elasticity and weak contraction. These results suggest that mutation of cTnT could both compromise filament structure and weaken contractile force.