Human Cardiomyocytes

Cardiomyocyte Isolation from Frozen Human Tissue

An overview of the isolation method is shown in Fig. 1A. Tissue was thawed and cut into 1 mm³ pieces while submerged in ice-cold wash solution (115 mM potassium gluconate, 2.5 mM potassium chloride (KCl), 5 mM potassium phosphate monobasic (KH₂PO₄), 2 mM magnesium sulfate (MgSO₄), 2 mM calcium chloride (CaCl₂), 30 mM sucrose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4). Tissue pieces were collected with a 70 μm cell strainer and washed three times with ice-cold wash solution. Tissue pieces were placed in digestion solution (300 Units/mL collagenase type 4, 5 Units/mL DNase I, and 1 mg/mL trypsin inhibitor in wash solution) in a 50 mL spinner flask at 37 °C. A total of three digestions were performed, each yielding a fraction of crude cardiomyocytes that was filtered through a 200 μm cell strainer. Crude cardiomyocytes from three digestions were combined, collected by centrifugation at 80 ×g for 5 min at 4 °C, and washed three times in ice-cold wash solution. Crude cardiomyocytes were further purified by removing immune cells and endothelial cells by negative selection. Isolated cardiomyocytes were assessed for percent troponin I (TNNI3) positivity by flow cytometry. Visual assessment of cardiomyocytes using immunofluorescent labeled cardiac actin was performed on a confocal laser scanning microscope.

Fig. 1. Cardiomyocyte isolation and purity assessment. A) Schematic overview of cardiomyocyte isolation workflow using cryopreserved human adult cardiac tissue. B) Representative confocal immunofluorescence images of isolated cardiomyocytes stained for cardiac actin (red). C) Flow cytometry assessment of isolated cells for TNNI3 positivity (Wojtkiewicz, M. et al., 2022).

Cardiomyocyte Contractility Measurement

With the ultimate goal of assessing both electrical (AP) as well as mechanical (contractility) drug-induced effects, we decided to focus our analysis on four parameters: TR90 (time to 90% relaxation; ms), incidence of AC (after-contraction), incidence of CE (contractility escape) and sarcomere shortening. TR90 is correlated to the duration of the cardiac AP and delays in AP repolarization are expected to be associated with extension of the TR90. Early-afterdepolarization (EAD) is an AP abnormality that results in a transient slope change of the AP during the repolarization phase. EAD is potentially of great relevance in the context of pro-arrhythmia risk assessment since this is believed to be the underlying cause of re-entrant arrhythmia. The mechanical equivalent of EAD electrical abnormality is an after-contraction, a transient change of slope in the contractility transient, typically in the later portion of the relaxation phase. Drugs that interfere with cardiac depolarization or in other ways suppress the generation of a cardiac AP, result in complete inhibition of the contractility transient, an event we refer to as CE. Therefore, changes in three parameters measured from contractility transients, TR90, AC and CE can provide useful information with regards to the drug-induced alterations of the electrical behavior of cardiac cells. In addition, changes in fractional sarcomere shortening provide direct measurement of inotropic effects in cardiomyocytes.

We recorded vehicle time-control data in four cardiomyocytes (one heart) using multiple additions of vehicle solution spaced by 4 min each, to mimic the experimental conditions that we were set to use with the test drugs. Cardiomyocytes exhibited stable behavior for the duration of the recordings, up to 20 min (Fig. 2). No AC or CE were observed and only a small, non-significant increase in TR90 was observed (Fig. 2A). Similarly, the measurements of sarcomere shortening in myocytes demonstrated good stability (Fig. 2B).

Fig. 2. Stability of contractility recordings over time in human cardiomyocytes. (A) Change in TR90 and % incidence of AC and CE induced by sequential additions of vehicle (V) in human cardiomyocytes at 1 Hz pacing frequency. (B) Vehicle effect curve for sarcomere shortening (Nguyen, Nathalie, et al., 2017).

Functional Characterization of Freshly Isolated hPCMs

First, we used patch clamping to characterize major membrane currents and their responses to specific inhibitors. Sodium currents were elicited by a series of depolarizing test potentials between −120 to +100 mV with 30 mV steps at a holding potential of −120 mV. The peak density of sodium current was at −20 mV, and the mean maximal peak I_Na normalized to cell capacitance was −39.27 ± 14.50 pA/pF (n = 10) (Fig. 3a). We also characterized the voltage-dependent steady-state activation and inactivation curves of sodium currents. The voltage at half activation (V_1/2) was −35.07 ± 0.87 mV (n = 10), with a mean slope factor (k) of 3.43 ± 0.76, whereas the V_1/2 for inactivation −72.13 ± 0.54 mV (n = 10), and the slope factor was 4.30 ± 0.47 (Fig. 3b, c). Time-dependent recovery of sodium currents from inactivation was assessed using a paired-pulse protocol. The mean recovery time constant of sodium currents was 3.83 ± 0.35 ms (n = 10) (Fig. 3d). Likewise, L-type Ca²⁺ currents (I_Ca,L) currents were elicited at potentials between −60 to +90 mV, with 30 mV steps at a holding potential of −80 mV. The I–V relationship showed that the peak density of calcium currents occurred at 30 mV, and the mean maximal peak I_Ca,L normalized to cell capacitance was −3.66 ± 0.98 pA/pF (n = 4) (Fig. 3e). We further characterized the current–voltage (I–V) relationships of two major potassium currents in freshly isolated hPCMs. For the 4-aminopyridine (4-AP)-sensitive, non-Ca²⁺-dependent transient outward potassium current (I_to), its current density averaged −0.13 ± 0.24 pA/pF at −40 mV, 2.17 ± 1.06 pA/pF at 0 mV, and 12.96 ± 4.32 pA/pF at +40 mV (Fig. 3f). The ultrarapid outward current I_kur is the other major repolarizing current in the human atrium. I_kur density averaged 0.24 ± 0.16 pA/pF at −40 mV, 2.00 ± 1.23 pA/pF at 0 mV, and 5.28 ± 3.39 pA/pF at +40 mV (Fig. 3g). Together, these data demonstrate proper ionic currents of isolated hPCMs.

To further validate the functionality of cardiomyocytes, we tested whether these currents responded correctly to specific channel inhibitors. To this end, we used tetrodotoxin (TTX), nifedipine, and 4-aminopyridine (4-AP) to respectively inhibit Na⁺ (I_Na), Ca²⁺ (I_Ca), and K⁺ (I_to) currents (Fig. 3h-j). The half-maximal inhibitory concentration (IC₅₀) values for TTX, nifedipine, and 4-AP were 16.24 μM, 55.39 nM, and 0.2563 mM, respectively, which were similar to reports in the literature (Fig. 3k-m). Taken together, isolated hPCMs exhibited normal electrophysiology, indicating their potential use as models to evaluate drugs that affect the electrical properties of cardiomyocytes.

We next used isoproterenol and norepinephrine to examine whether cells responded to neurohormonal stimulation. Both stimuli caused time- and dose-dependent increases in the phosphorylation of phospholamban, an important regulator of calcium handling in cardiomyocytes (Fig. 3n), demonstrating that cells retained functional adrenergic signaling following isolation.

Fig. 3. Functional integrity of freshly isolated hPCMs (Zhou, Bingying, et al., 2022).