PMC

ATP-activated currents (I_ATP) in CPCeP (n = 9), CPCeP attached to NRCM (n = 13), and NRCM alone (n = 9). A: Representative traces of I_ATP in individual CPCeP and NRCM. B: Averages of maximum I_ATP current density in each group. Cells were voltage-clamped to −80 mV for 8 s and exposed to 100 µM ATP for 1 s.

Sodium currents (I_Na) in CPCeP attached to NRCM, in NRCM alone, and in NRCM attached to CPCeP. A: Sample traces of membrane current activated by step-depolarization from a holding potential of −80 mV. B: Average time-to-peak values of I_Na measured at the peak value of the I–V relations. C: I–V relations of the density of I_Na. D: Average peak values I_Na in each group.

Immunostaining of connexin 43 (Cx43, red) in eGFP-expressing CPCeP (green) cultured alone (A–D) or together with NRCM (E–G). Details of panels A and E are shown in panels B and F, respectively, where the red color corresponding to Cx43 staining is accentuated by being shown within the outlines of the CPCeP (dashed white lines) without the green color that identified these cells. Clustering of Cx43 (arrows) on the boundaries between cells is illustrated in panels C (CPCeP–CPCeP), D (CPCeP-*) and G (CPCeP–NRCM; arrows). Scale bars in white are 10 µm.

Na⁺–Ca²⁺ exchanger currents (I_NCX) in neonatal rat cardiomyocyte (NRCM, n = 6), cardiac progenitor cell engineered to express eGFP and Pim-1 kinase (CPCeP) cultured alone (n = 9) or attached (n = 7) to NRCM. A and B: Representative current traces (A) and I–V relations (B) measured with ramp-clamp protocol (inset) in the absence (black) and presence of 5 mM Ni²⁺ (red). C: Bar graph showing the densities of the Ni²⁺-sensitive I_NCX at +80 and −120 mV.

Paracrine effects of neonatal rat cardiomyocyte (NRCM) on I_Ca in cardiac progenitor cell engineered to express eGFP and Pim-1 kinase (CPCeP). A: Percentile distributions of the magnitudes of I_Ca in responsive CPCeP according to the conditions of the culturing process (regular medium vs. co-culture with NRCM vs. conditioned medium) and its duration (1–3 days or 4–7 days). B: Average I_Ca in responsive CPCeP (black symbols) and CPC (red symbols). C: Fraction of responsive CPCeP and CPC. The numbers next to each data indicate the number of responsive cells producing significant I_Ca (>0.4 pA/pF; B) and this number divided by the total number of voltage-clamped cells (C).

Calcium currents (l_Ca) in cardiac progenitor cell engineered to express eGFP and Pim-1 kinase (CPCeP, n = 8) attached to neonatal rat cardiomyocyte (NRCM) and in NRCM without (n = 6) or with (n = 3) attachments to CPCeP. A: Representative traces of I_Ca using voltage-step depolarizations to different test potentials from a holding potential of −40 mV. B: Average time-to-peak values of I_Ca. C: Current–voltage (I–V) relations for I_Ca normalized relative to the membrane capacitance. D: Average values of peak I_Ca measured at 0 mV in each group. Stars indicate significance levels (*p<0.05, **p<0.01) while n indicates the number of voltage-clamped cells.

Prevalence membrane currents (I_Ca, I_Na, I_ATP, I_NCX) in voltage-clamped CPCeP cultured alone (panel D, red bars) or with NRCM (panel D, hatched bars). A: Co-cultured cells with eGFP-labeled CPCeP showing green fluorescence superimposed on the bright field image also showing associated NRCM and the voltage-clamp pipette (µ). B and C: Separately cultured CPCP generated no detectable I_Ca when tested with a voltage-clamp protocol consisting of repeated step depolarizations of increasing amplitude (panel B), but generated a nifedipine-sensitive inward I_Ca (arrow) during brief ramp depolarizations (panel C). D: Prevalence of responsive cells. The numbers at the top of each bar (n₁/n₂) indicate the number of cells with clearly detectable current (n₁) out of the total number of tested cells (n₂). I_Ca in separately cultured CPCeP was probed using both step- (STEP) and ramp-(RAMP) protocols. I_ATP was measured at a fixed holding potential (HOLD), while I_NXC was recorded with a repolarizing ramp (RAMP).

Calcium transients in co-cultured CPCeP and NRCM. Panels A–C show details of the Ca²⁺ transients elicited by 10 mM caffeine (A), 100 mM KCl (B) and 100 µM ATP (C). The graphs on the left show the time course of the Ca²⁺ signals (F/F₀) measured in color-coded regions of interest (panel E) corresponding to NRCM (red and orange) and CPCeP (light blue, dark blue, green, olive, purple, violet). The sample frames in panels A–C show ratiometric images recorded during (1) and after the interventions (2) at the times indicated in the graphs. D: Baseline fluorescence (F₀). F: Average Ca²⁺ transients (F/F₀-1) evoked by caffeine (Caff), KCl-depolarization (KCl) and ATP (ATP) in multiple NRCM (red bars) and CPCeP (blue bars). The cells were imaged confocally at a frame rate of 30 Hz after incubation with Fluo-4 AM.

Configuration of CPCeP examined separately and in co-culture with NRCM. A–C: Images of single CPCeP cultured alone for 1–2 days (A) and 5–7 days (B) and with NRCM for 5 days (C). In panels A and B, the green fluorescence from eGFP is superimposed on the bright field images. D: Capacitive membrane currents of an isolated voltage-clamped CPCeP before (black) and after (red) decoupling of an adjacent NRCM. E: Average values of the total membrane capacitance measured in different voltage-clamp configurations. The icons, that are also used in the following figs., show a CPCeP at the end of a patch electrode, the same configuration with an attached cardiomyocyte (PCPeP + NRCM), and an isolated voltage-clamped cardiomyocyte (NRCM). F and G: Transfer of dye from a voltage-clamped PCPeP (red and orange regions of interest) to an attached NRCM (blue and green regions of interest). F: Regions of interest (ROI) and sample frames measured at 4 and 14 min. G: Time course of normalized fluorescence intensity in the regions of interest.