Patient-Derived Cardiomyocytes Separate GPCR Signaling from Functional Response
A four-donor study compares nuclear and cytoplasmic signaling with calcium, electrical and impedance measurements. Distinct responses in DCM-derived cells show why increased signaling cannot alone establish improved cardiac-cell function.
Four Donor Lines Provide an Exploratory Comparison
Researchers at McGill University and the McGill University Health Centre generated induced pluripotent stem cell-derived cardiomyocytes from two healthy controls and two patients with dilated cardiomyopathy (DCM). They compared responses to G protein-coupled receptor (GPCR) ligands at molecular and functional levels, with the aim of identifying individual cellular profiles relevant to drug research.
The patient cases had different presumed exposure-related causes, and no mutations in known DCM-associated genes were identified. The investigators reprogrammed blood-derived cells, verified pluripotency and chromosomal normality, and confirmed cardiomyocyte differentiation through beating and marker expression. The reported protocol typically yielded approximately 85% cardiomyocyte purity. Each line represented one donor, so the study remained a four-person proof of concept.
Subcellular Signaling Reveals Different Response Patterns
Biosensors measured protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) activity separately in the nucleus and cytoplasm. The ligand panel probed angiotensin, adrenergic and endothelin receptors, with pathway activators used as experimental references. Measuring downstream activity avoided requiring receptor overexpression and allowed the same culture to be examined at single-cell resolution.
The first patient-derived line showed a comparatively strong cytoplasmic PKA response to phenylephrine, an α1-adrenergic agonist, and elevated nuclear ERK response to endothelin-1. In the second patient line, forskolin indicated lower overall PKA activation capacity than in its matched control. Adrenergic responses also differed between patient lines, while many comparisons with the corresponding healthy line were not statistically significant.
The angiotensin ligands produced no PKA response in the tested cells, and most ERK responses were small or variable. Carvedilol did not significantly change PKA in either compartment. These null findings constrain which receptor–effector relationships the assay detected under its conditions.
Single-cell clustering showed that a culture-average signal could reflect a small highly responsive subpopulation together with many weakly responding cells. Cluster proportions changed with the ligand and donor. This analysis added information about cellular heterogeneity that was obscured by a single average.
Calcium, Electrical and Impedance Assays Do Not Always Align
A myofilament-localized calcium reporter measured spontaneous transients before and after 10 μM isoproterenol. Baseline rates were roughly 40 beats per minute across the lines. Most showed a rate increase, whereas the second patient line showed little change. Isoproterenol did not significantly change transient amplitude, upstroke velocity or area under the curve in the reported within-line comparisons.
Extracellular field-potential recordings produced a different frequency result: isoproterenol increased the rate by roughly 20–30 beats per minute in all four lines. The authors discuss assay conditions, including pacing during concurrent impedance recordings, as a possible contribution to the second patient line’s differing responses. The calcium experiment measured spontaneous events, while impedance was measured with 1-Hz pacing.
Field-potential amplitude was lower in the patient-derived cultures, although only one control line was significantly higher than both patient lines. Neither field-potential amplitude nor corrected field-potential duration changed significantly after isoproterenol. Impedance measurements supplied contraction-related amplitude and timing information; these quantities do not directly measure absolute force. The first patient line had particularly low impedance amplitude.
A Stronger Molecular Signal Does Not Guarantee a Functional Gain
Dose-response experiments made the signaling–function distinction concrete. Phenylephrine produced no apparent increase in beat rate or impedance amplitude, including in the first patient line with its elevated PKA response. The authors propose that signaling strength, compartmentalization or coupling could explain this mismatch, but the experiment does not select one explanation.
Isoproterenol increased beat rate across the lines with limited changes in impedance amplitude. Endothelin-1 also increased rate despite little PKA activity, while impedance amplitude tended to decrease. These endpoint-specific responses support measuring functional consequences directly when a signaling pathway is being considered as a therapeutic target.
The investigators propose adding maturation-marker imaging and registering individual cells across successive measurements, then linking the resulting profiles with clinical data. Larger cohorts, including patients with known genetic etiologies, would test whether the signaling/function patterns identify reproducible subgroups. The current four-donor design demonstrates experimental feasibility and individual variation.