K2p3.1 protein is expressed as a transmural gradient across the rat left ventricular free wall

Figures

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  Figure 1. Single epicardial and endocardial myocytes immunofluorescent label of K2p3.1 protein. A, Illustrative example high‐resolution images of K2p3.1 protein labelling in single myocytes from the subepicardial (left) and subendocardial (right) regions of the left ventricle. B, Single myocyte cross‐sectional area for cells from each region. C, Mean total immunofluorescence of labelled K2p3.1 protein per myocyte. D, The mean density of immunofluorescence of labelled K2p3.1 protein (each panel shows means ± SEM; n = 92 cells per group; bars denote significant differences by t test; *P < 0.001)

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  Figure 2. Transmural gradient of immunofluorescent‐labelled K2p3.1 protein expressed across the left ventricular free wall. A, Illustrative high‐resolution image of myocytes within the left ventricular wall with K2p3.1 protein labelled. B, Example of K2p3.1 protein labelling in a cross‐section of the left ventricular free wall. B‐C, A white dashed line is oriented perpendicular to the epicardial surface and is drawn to the endocardial surface in (B), which indicates the line used to determine a transmural profile of immunofluorescent labelled K2p3.1 protein in (C). C, A profile of fluorescence from an example tissue section shows the increase in immunofluorescent‐labelled K2p3.1 protein from the epicardial surface to the endocardial surface across the left ventricular free wall. D, Intensity of labelled K2p3.1 protein (n = 49) compared at 20% distance intervals across the left ventricular wall. Mean values were normalized to signal in the most epicardial interval (0%‐20% distance across the left ventricular wall). Data are shown as means ± SEM; n = 49 sections per group; one‐way analysis of variance with Holm‐Sidak comparisons *P < 0.001

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  Figure 3. Quantification of K2p3.1 protein and mRNA expression. A, An example of a Western blot showing the identified specific single band labelled for K2p3.1 protein (and desmin protein band for normalization). Significantly more K2p3.1 protein was expressed in tissue from the subendocardial than the subepicardial layer (mean ± SEM; n = 8; Student's t test *P < 0.0001). B, No significant difference in the amount of K2p3.1 mRNA as expressed relative to GAPDH mRNA was identified between regions (mean ± SEM; n = 10). GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA

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  Figure 4. K2p3.1 Current‐voltage relationship in the left ventricular free wall. Subepicardial and subendocardial layer–derived myocytes were whole‐cell voltage‐clamped. A, Cells were voltage‐ramped from −50 to 30 mV over 6 seconds with a holding potential of −10 mV (upper line, black), whole‐cell currents were recorded (lower line, red). B, Current‐voltage relationship in the presence (met) and absence (control) of a specific K2p3.1 inhibitor methanandamide. C, Example of the methanandamide‐sensitive current‐voltage relationship from a single ventricular myocyte. D, Current‐voltage relationship from epicardial and endocardial myocytes. E, Average currents at 30 mV, taken as the peak outward current, were shown in the endocardial myocyte to be double that measured in the epicardial myocyte (Students t test, *P < 0.001; Epi n = 7; Endo n = 6)