Background:
Coordinated, efficient cardiac muscle contraction requires precise orchestration of intracellular Ca²⁺ signals in individual cardiomyocytes. However, quantitative observations reveal pronounced cellular heterogeneity within cell populations – both in intracellular Ca²⁺ dynamics (see Front Physiol. 2015;5:517) and in the contractility of individual cardiac muscle cells. This functional diversity could critically influence the efficiency of cardiac excitation-contraction coupling and may represent an underestimated mechanism in both the physiological behavior of cardiomyocytes and the pathophysiology of cardiac diseases. However, it remains unclear to what extent this heterogeneity is reflected in the contractile function of isolated cells and how it changes under physiological stress, such as β-adrenergic stimulation. The origin of heterogeneous functional signaling responses within the β-adrenergic signaling cascade is also unknown.
Methods:
Ventricular cardiomyocytes were isolated from Langendorff-perfused hearts of nine-week-old male Wistar rats. Ratiometric fluorescence microscopy (Cytocypher) was used to record intracellular Ca²⁺ transients and sarcomere shortening under basal conditions and after stimulation with isoproterenol (β-agonist) and forskolin (adenylyl cyclase agonist) in large cell populations. Analyzed parameters included contraction dynamics (sarcomere shortening), contraction/relaxation times, Ca²⁺ amplitude, and temporal Ca²⁺ kinetics.
Results:
Intercellular differences in contractility and Ca²⁺ dynamics were already observed under basal conditions. Isoproterenol caused significantly stronger sarcomere shortening (p < 0.001) and increased systolic [Ca²⁺] (p = 0.0079), but also heightened intercellular variability in Ca²⁺ parameters. For example, the Ca²⁺ amplitude range spanned 1.3265–2.3052 F340/F385 in the 0.1 µM forskolin, 1 µM forskolin, and control groups, compared to 18.0249 F340/F385 in the isoproterenol group. Kernel smoothing density plots visualized this variable data distribution across groups.
Forskolin at 1 µM (p < 0.001) and 10 µM (p < 0.001) also induced significant sarcomere shortening. Compared to isoproterenol, 10 µM forskolin produced stronger sarcomere shortening with less Ca²⁺ dynamic heterogeneity.
Conclusion:
The results suggest functional heterogeneity within ventricular cardiomyocyte populations, indicating functional subgroups. Cells with weak responses to low forskolin concentrations retained cellular reserves recruitable by higher concentrations, leading to enhanced responses.
The greater heterogeneity under isoproterenol versus forskolin implies heterogeneity factors in pathways upstream of adenylyl cyclase. This variability may stem from differences in β-adrenergic receptor expression/sensitivity or G protein-mediated signal transduction. The observed functional heterogeneity under physiological stress could represent a previously overlooked modality influencing myocardial adaptation in health and disease.
