Preferential Expression of Ca2+-Stimulable Adenylyl Cyclase III in the Supraventricular Area, including Arrhythmogenic Pulmonary Vein of the Rat Heart

Figures

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  Figure 1. Macroscopic view of the preparation and genetic RT-PCR screening for adenylyl cyclase (AC) superfamily members. (A) A stereomicroscopic snapshot of the cardiopulmonary preparation focusing on a left pulmonary vein (PV). To visualize the veins, fresh blood was left inside. The PV extends from the left atrium (LA) and visibly branches into the left superior lung lobe. The post caval lobe of the lung is behind the left PV. The branches from the right PV are out of focus and cannot be fully seen in this view. A part of the left ventricular (LV) mass is observed at the bottom. Scale bar: 5 mm with 1 mm increments. (B) Quantitative RT-PCR for AC1–8. By fitting each quantitative cycle to a standard linear curve, the abundance of each reverse-transcribed cDNA is measured quantitatively. The exogenous standard curves are prepared by RT-PCR of diluted RNA extracted from the right atrium. Accordingly, the vertical axis corresponds to the same amount of standard cDNA. The ordinary one-way analysis of variance indicates that expression levels significantly vary from isotype to isotype (p < 0.0001). Tukey’s multiple comparisons test identified statistically significant differences. AC1, AC3,…

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  Figure 2. Immunohistochemistry of a left-sided rat heart for adenylyl cyclase AC1, AC2, AC3, AC6, and AC8. (A) Masson’s trichrome staining in a left-sided heart from a male Wistar rat. Muscle, collagen-enriched tissue, and elastin-enriched fibers are stained purple, turquoise, and dark blue, respectively. The cardiopulmonary regions are indicated by bold letters. LA, left atrium; LV, left ventricle; PV, pulmonary vein; RV, right ventricle. (B) Immunohistochemistry for AC6. AC6 is known as a heart subtype of AC and presents a positive control with solid staining (brown). (C–F) Immunohistochemistry for AC1–3 and AC8. AC1 and AC8 are both moderately positive in (C,F). (D) AC2 is almost negative in the heart, while the lung epithelium is positively stained. (E) AC3 expression is restricted to the LA and PV. Scale bars: 5 mm.

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  Figure 3. Immunohistochemistry of a right-sided rat heart for adenylyl cyclase AC3. (A) Masson’s trichrome staining. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle, SAN, sinoatrial node; SVC, supra vena cava. (B) Immunohistochemistry for AC3. The supraventricular area except the SAN is stained positive. Scale bars: 5 mm.

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  Figure 4. Immunostaining of adenylyl cyclase AC3. (A) Supraventricular regions with Masson’s trichrome staining (Aa) and immunostaining for AC3 (Ab). The ventricular septum is visible beneath the mitral valve on the lower left, a connective tissue-enriched thin structure. The lung is on the upper right. The tentative boundary line between the left atrium (LA) and pulmonary vein (PV) is indicated by a dotted line. (B) Intercaval regions with Masson’s trichrome staining (Ba) and AC3 (Bb) immunostaining. The sinoatrial node (SAN) is defined as a myocardial region surrounded by collagen-enriched connective tissues and contains the SAN artery. The artery is observed as circular cavities. The area indicated by the dotted line is the SAN. The superior vena cava is located immediately above the SAN to the right.

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  Figure 5. Immunocytochemistry for adenylyl cyclase AC3 in the myocytes of (A) isolated left atrium (LA), (B) left ventricle (LV), and (C) pulmonary vein (PV). α-actinin (green, all top panels) localized to the z-line of cardiomyocytes, indicating the position of the t-tubules. AC3 staining (red, all middle panels) revealed transverse striations in LV and PV myocytes, unlike in LA myocytes. AC3 is strongly expressed along the cell contour in LA myocytes. The bottom panels display the overlay of AC3 on α-actinin (yellow-orange). The white bars in the bottom panel indicate 10 μm. (D,E) and (F) display the profiles of α-actin and AC3 staining intensities in (A–C), and the cross-correlation between both intensity profiles, respectively. The area to be intensity-profiled is chosen from the rectangular box in the bottom panel of (A–C), all of the same size. The cross-correlation waveforms indicate that the intensities of actin and AC3 are synchronized, suggesting that they are localized near each other [11,32]. It should be noted that the nuclei of LA and LV are stained with AC3. Neither the laser intensity of the microscope nor the gain during imaging is increased in these imaging compared to PV…

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  Figure 6. Ca2+-dependent effects of β-adrenergic stimulation on L-type Ca2+ currents in three types of cardiomyocytes. As shown in the inset of (A), the Ca2+ current is induced by a depolarizing pulse to 0 mV for 500 ms, followed by a depolarizing pulse to −40 mV for 80 ms to inactivate the Na+ current. The L-type Ca2+ current is amplified by 1 μM isoproterenol (Iso.), a β-adrenergic receptor-selective agonist. The strength of the β-adrenergic stimulation is evaluated by the rate of amplification of the current amplitudes. It was observed whether the absence or presence of intracellular Ca2+ changed the strength of the β-adrenergic reaction. In Figure 8A,C,E, three stable sweeps are superimposed under control conditions (Cont., black) or Iso. conditions (red). Inserted time and current scales indicate 50 ms and 5 pA/pF, respectively. Dashed lines indicate zero current level. Intracellular Ca2+ is chelated by 5 mM EGTA under the Ca2+-free condition, while the internal solution contains 0.1 mM Ca2+ in the other. (A) Representative data from pulmonary vein (PV) cardiomyocytes. A statistical summary of an increase in calcium current by Iso. for each condition is shown in (B). (C)…

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  Figure 7. Effect of SQ22536 (SQ) on L-type Ca2+ current in pulmonary vein (PV) cardiomyocytes. The response of the Ca2+ current was recorded after 5 μM SQ application for approximately 2 min. The pulse protocol is inserted in (A). Intracellular Ca2+ is chelated by 5 mM EGTA in (A), while the internal solution contains 0.1 mM Ca2+ in (B). Dashed lines indicate zero current level. The rate of change in the current amplitudes for each condition is summarized in (C). Of note, under Ca2+-rich conditions, a Ca2+-dependent inward current indicated by an arrow in (B) is always recorded. This current is estimated to be of Na+-Ca2+ exchanger origin.

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  Figure 8. Effects of SQ22536 (SQ) on the norepinephrine (NE)-induced automaticity of pulmonary vein (PV). (A) An example of NE-induced spontaneous automaticity. NE 10 µM evoked a series of action potentials in isolated rat PV cardiomyocytes. The action potentials recorded at a, b, and c are displayed in an expanded time scale below. Inserted time and voltage scales indicate 50 s and 20 mV, respectively. The time scale in the expanded view indicates 500 ms. The induced automaticity was uninhibited by 5 µM SQ. (B) The induced automaticity was reversibly suppressed by 100 µM SQ. Inserted time and voltage scales indicate 50 s and 20 mV, respectively. The time course of the inhibition is expanded in (C). The sporadic depolarizations corresponding to Ca2+ release were gradually attenuated (arrowheads). Similar results were confirmed four times (n = 4, for each). The timing and duration of drug applications are indicated by horizontal bars. A dashed line indicates the baseline.