Evidence of intercellular coupling between co-cultured adult rabbit ventricular myocytes and myofibroblasts

Abstract

Intercellular coupling between ventricular myocytes and myofibroblasts was studied by co-culturing adult rabbit ventricular myocytes with previously prepared layers of cardiac myofibroblasts. Intercellular coupling was examined by: (i) tracking the movement of the fluorescent dye calcein; (ii) immunostaining for connexin 43 (Cx43); and (iii) measurement of intracellular [Ca2+] ([Ca2+]i). The effects of stimulating ventricular myocytes on the underlying myofibroblasts was examined by confocal measurements of [Ca2+]i using fluo-3. When ventricular myocytes were preloaded with calcein and co-cultured with myofibroblasts for 24 h, calcein fluorescence was detected in 52+/-4% (n=8 co-cultures) of surrounding myofibroblasts. Treatment with the gap junction uncoupler heptanol significantly reduced the movement of calcein (12+/-3%, n=6 co-cultures). Immunostaining showed expression of Cx43 in co-cultured myofibroblasts and myocytes. Field stimulation of ventricular myocytes co-cultured with myofibroblasts increased myofibroblast [Ca2+]i, no response was observed after treatment with heptanol or stimulation of fibroblasts in the absence of ventricular myocytes. Action potential parameters of ventricular myocytes in co-culture were similar to control values. However, application of the hormone sphingosine-1-phosphate (S-1-P) to the co-culture caused a depolarization of ventricular myocytes to approximately -20 mV. Sphingosine-1-phosphate had no effect on ventricular myocytes alone. Voltage-clamp measurements of isolated myofibroblasts indicated that S-1-P activated a significant quasi-linear current with a reversal potential of approximately -40 mV. In conclusion, this study shows that stimulation of the ventricular myocyte influences the intracellular Ca2+ of the linked myofibroblast via connexons. These intercellular links also allow the myofibroblasts to influence the electrical activity of the myocyte. This work indicates the nature of the gap junction-mediated bi-directional interactions that occur between ventricular myocyte and myofibroblast.

Figure 2

C × 43 staining in co-cultured myocytes and fibroblasts A, immunostaining for Cx43 was robust in myofibroblasts and ventricular myocytes maintained in co-culture for 24 h. Aa, immunofluorescent image showing FITC fluorescence, with Cx43 staining shown as punctate green spots. Ab, light micrograph of co-culture. Arrows indicate individual myocytes lying on a confluent field of myofibroblasts. Scale bar represnts 15 μm. Appropriate controls showed no non-specific staining. The areas indicated by the white dashed lines are shown expanded in Ba and b. The outer border of the ventricular myocyte is highlighted in both panels.

Figure 1

Evidence of calcein dye-coupling between adult rabbit myofibroblasts and ventricular myocytes in co-culture Aa, representative transmitted light image of a myocyte pre-incubated with calcein AM following 24 h co-culture on a layer of myofibroblasts. Ab and c, calcein fluorescent images of an optical section through the middle of the myocyte (Ab) and an optical section from 10 μm below this (Ac), which contains only myofibroblasts. The position of the overlying ventricular myocyte is indicated with a grey outline. Scale bars represent 20 μm. B, mean ±s.e.m. (n) percentage of myofibroblasts exhibiting calcein fluorescence following 24 h co-culture under control conditions (black bar), with 2 mmol l⁻¹ heptanol present in the culture media (grey bar), and within 4 h after addition of ventricular myocytes (right).

Figure 3

Variation in fluo-3 fluorescence in myofibroblasts co-cultured with adult rabbit ventricular ventricular myocytes for 24 h Aa, light micrograph showing a single myocyte after 24 h co-culture with myofibroblasts. A composite image of fluo-3 fluorescence is shown in Ab, in which ‘M’ refers to the myocyte. The three myofibroblasts which were recorded from are identified. B, fluo-3 intensity in the myocyte (black) and in individual myofibroblasts (grey lines) within the 215 μm² area studied. C, average change in signal in 40 myofibroblasts associated with a single myocyte within a different 215 μm² area. D, response of myofibroblasts to exogenous ATP (1 mm). The time periods over which the myocytes were field stimulated are denoted in B, C and D.

Figure 4

Effects of 20 nmol l⁻¹ sphingosine-1-phosphate (S-1-P) on action potential characteristics Application of 20 nmol l⁻¹ S-1-P during 1 Hz pacing depolarized and blocked excitability in a representative myocyte co-cultured with myofibroblasts (A, left trace, and B). The S-1-P-induced depolarization and loss of excitability were reversible following 10 min washout (A, right trace).

Figure 7

Application of S-1-P and depolarization did not affect myofibroblast [Ca²⁺]_i A, myofibroblast membrane potential (in mV). Myofibroblasts in monoculture were depolarized at a rate of 1 Hz from −90 to 30 mV for 300 ms, mimicking the parameters of the average ventricular myocyte action potential. The time between sections is approximately 90 s. B, representative trace of myofibroblast current evoked by the step depolarization depicted in A, and with S-1-P application (20 nmol l⁻¹; right trace). C, representative trace of myofibroblast fura-2 ratio prior to depolarization (left section), during ‘mock action potentials’ (middle section), and with 20 nmol l⁻¹ S-1-P present (right section). Neither depolarization nor application of S-1-P affected myofibroblast fura-2 ratio.

Figure 6

Concentration dependence of the S-1-P-induced current change in myofibroblasts A, representative traces from a monocultured myofibroblast voltage-clamped at −90 mV and then stepped repetitively to 30 mV for 300 ms at 1 Hz. Current changes recorded under control conditions (black line) and in response to increasing concentrations of S-1-P (grey lines) are shown. The response of a representative myofibroblast to S-1-P over the S-1-P concentration range (^= 30 mV; •=−140 mV) is shown in B, while mean increases in current are shown in C (means ±s.e.m., n = 11).

Figure 5

Sphingosine-1-phosphate activated a transmembrane ionic current in myofibroblasts Application of 20 nmol l⁻¹ S-1-P evoked an increase in the whole-cell currents in voltage-clamped myofibroblasts. A, representative current–voltage relationship under control conditions (^) and after 1–2 min 20 nmol l⁻¹ S-1-P application (•). The inset panel is the difference current. Application of vehicle did not cause any change in myofibroblast current. B, transmembrane ion currents under control conditions (open bars) and evoked by 20 nmol l⁻¹ S-1-P (black bars) at −90 (left) and 30 mV (right, means ±s.e.m., n = 13).

Figure 8

Injection of a rectangular depolarizing current waveform depolarized and blocked excitability of ventricular myocytes in monoculture In 24 h monocultures of ventricular myocytes, action potentials were evoked at 1 Hz by injection of 1.2 × threshold 10 ms current steps (A, top trace). A long depolarizing injection of current was then applied until diastolic membrane potential failed to repolarize and excitability was lost (A, bottom trace). As shown in B, the range of injected currents over which ventricular myocyte membrane potential remained depolarized was very narrow. Both panels show representative data from single ventricular myocytes. On average, injection of 0.17 ± 0.02 nA (n = 22) of current was associated with a sustained diastolic depolarization from −72 ± 1 to −11 ± 3 mV (n = 22). At this membrane potential, a loss of excitability in the monocultured myocyte was observed.