Cardiomyocyte sulfonylurea receptor 2-KATP channel mediates cardioprotection and ST segment elevation

Abstract

Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.

Fig. 1.

Restoration of cardiomyocyte sulfonylurea receptor 2 (SUR2)A protein in the SUR2 null heart. Top left: schematic map of MLC-2v SUR2A transgene construct. The 270-bp MLC-2v promoter (black box) is immediately proximal to the MLC2 gene and drives cardiomyocyte-specific expression. The SUR2A variant of SUR2 contains exon 39 and is the primary SUR2 splice variant expressed in heart and skeletal muscle. “Ter” represents the bovine growth hormone terminator. Restriction enzyme sites are indicated: X, XhoI; N, NotI; C, SacII; S, SalI. MLC2A transgene-bearing mice were bred to SUR2 null mice to restore cardiomyocyte SUR2-containing ATP-sensitive potassium (K_ATP) channels. Immunofluorescence microscopy of heart sections from wild-type (WT) control (top right), MLC2A (bottom left), and SUR2 null (bottom right) mice is also shown. All sections were stained with anti-SUR2 antibodies to visualize SUR2 (green) and DAPI (blue) to visualize nuclei. SUR2 staining was evident in control and MLC2A hearts but not in hearts from SUR2 null mice. Scale bars = 50 μm in all micrographs.

Fig. 2.

Restoration of cardiomyocyte K_ATP channel activity in MLC2A mice. Inside-out patches of K_ATP currents were determined from cardiomyocytes isolated from hearts of each genotype. A: representative examples of multichannel excised patch recordings of K_ATP currents at a holding potential of 0 mV. Open channel currents are represented as downward deflections. The closed level is marked as 0 pA. K_ATP currents were measured in ventricular myocytes isolated from litter-matched control, SUR2 null, and MLC2A mice, with records taken before, during, and after 1 mM ATP administration as shown by the bars. B: % of patches that contained at least some K_ATP currents in ventricular myocytes isolated from 4 control, 3 SUR2 null, and 3 MLC2A mice. The actual number of patches with current/total number of patches is shown above each bar. C: summary of mean amplitude of K_ATP currents for those patches containing currents in the control, SUR2 null, and MLC2A cells. *P < 0.001 vs. SUR2 null. ns, Not significant.

Fig. 3.

Cardiomyocyte expression of SUR2A in MLC2A mice results in smaller infarct sizes. Isolated hearts from SUR2 null (n = 13) and MLC2A (n = 7) mice were exposed to 40 min of “no-flow” ischemia via a standard Langendorff setup. After 60 min of reperfusion, hearts were sectioned and stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) to visualize infarct size. Restoration of cardiomyocyte SUR2A expression resulted in increased resistance to ischemia [area at risk (AAR)]. *P < 0.05.

Fig. 4.

MLC2A mice exhibit improved cardiac function after ischemia compared with SUR2 null mice. Left ventricular developed pressure (LVdevP) was measured with a pressure-sensing catheter attached to a fluid-filled balloon placed inside the left ventricle. Baseline parameters were similar for all cohorts. A: isolated hearts from MLC2A mice (n = 5) exhibited increased LVdevP after ischemia compared with hearts from SUR2 null mice (n = 10). *P < 0.05. B: systolic pressure was similar between cohorts. C: diastolic pressure was significantly reduced in isolated hearts from MLC2A mice compared with SUR2 null and MLC2A Line 8 isolated hearts. *P < 0.01 vs. SUR2 null baseline LVdevP; ^#P < 0.001 vs. SUR2 null.

Fig. 5.

ST segment elevation episodes are reduced in frequency in MLC2A compared with SUR2 null mice. Conscious telemetric ECG recording was conducted on SUR2 null (n = 9) and MLC2A (n = 5) cohorts for 48-h intervals. A: examples of ST segment elevation are shown from an episode lasting ∼1 min. A baseline tracing just before an ST segment episode is shown in 1. This was followed by an ST segment episode shown in 2. Recovery following the ST segment elevation is shown in 3. B: T-wave amplitude was plotted as an indicator of ST segment elevation over the time frame that includes the data shown in A, where 1, 2, and 3 correlate with those same time points in A. The T-wave amplitude is increased at point 2, reflecting ST segment elevation. C: heart rate [in beats/min (bpm)] was plotted for the same time window as in A and B, showing that heart rate declines during ST segment elevation, as we previously noted (5). D: we used an algorithm to measure T-wave amplitude and found that that MLC2A rescued mice had significantly less ST segment elevation than SUR2 null mice, reflecting a reduction in frequency and duration of episodes. The observed reduction of coronary vasospasm in MLC2A mice was significant (P < 0.05).