Pharmacologic ROMK Inhibition Protects Against Myocardial Ischemia Reperfusion Injury

Figures

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  Figure 1. The effect of ROMK inhibition during in vivo IR injury in mice. (A) IR injury protocol that subjected mice to 45 min of ischemia and 3 days of reperfusion. The ROMK inhibitor, Compound A, or vehicle was given prior to the ischemic insult and throughout the reperfusion period. (B) Representative images of heart slices stained with Evans blue and TTC after IR injury. The yellow dashed line represents the area at risk, and the white solid line represents the infarct area. (C) Cumulative data of % area at risk and infarct size after IR injury. (D) Survival of mice treated with ROMK inhibition vs. vehicle. Like the ROMK inhibitor treated group, there was no mortality in the Sham arm (not shown). (E) Echocardiography after IR injury. LVID; d, left ventricular end diastolic dimension; LVID;s, left ventricular end systolic dimension; EF, ejection fraction; FS, fractional shortening. N = 14 mice for vehicle-treated; N = 12 mice for ROMK inhibitor-treated. # p < 0.05 vs. sham. * p < 0.05 vs. vehicle. ** p < 0.01 vs. vehicle. Comparisons in (B) were made with Student’s t-test. One-way ANOVA with the post hoc Tukey HSD test was performed to assess differences between groups in…

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  Figure 2. Hemodynamic response during ex vivo IR injury between vehicle and ROMK inhibitor-perfused mouse hearts with and without ischemic preconditioning. (A) IR and IPC/IR protocol. Cumulative traces of isolated heart hemodynamic recovery in (B) LVDP, (C) +dp/dt, and (D) −dp/dt. N = 7 mice/group; * p < 0.001 vehicle IR vs. ROMK inhibitor IR; † p < 0.01, vehicle IR vs. vehicle IPC/IR; ‡ p < 0.001, vehicle IR vs. vehicle IPC/IR; | p < 0.05 vehicle IPC/IR vs. ROMK inhibitor IPC/IR; Ï p < 0.001 vehicle IPC/IR vs. ROMK inhibitor IPC/IR; # p < 0.01 ROMK inhibitor IR vs. ROMK inhibitor IPC/IR, ¶ p < 0.001 ROMK inhibitor IR vs. ROMK inhibitor IPC/IR. Two-way ANOVA was performed to assess differences between groups.

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  Figure 3. Infarct size after ex vivo IR injury (45 min ischemia, 60 min of reperfusion) between vehicle and ROMK inhibitor-perfused mouse hearts with and without ischemic preconditioning. (A) Representative heart images. (B) Cumulative % infarct data. N = 7 mice/group; * p < 0.05. One-way ANOVA was performed with the post hoc Tukey HSD test to assess differences between groups.

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  Figure 4. The effect of ROMK inhibition on FAD autofluorescence. (A) Cumulative traces of FAD autofluorescence from isolated murine cardiomyocytes pretreated with and without a ROMK inhibitor subsequently perfused with 1 μM FCCP followed by 1 mM NaCN. (B) Cumulative data of baseline FAD autofluorescence between vehicle and ROMK-treated cells. (C) Representative images of isolated cardiomyocytes treated with and without a ROMK inhibitor perfused with 100 µM Diazoxide followed by 1 μM FCCP and 1 mM NaCN; scale bar = 50 μm. (D,E) Cumulative traces of FAD autofluorescence in vehicle or ROMK inhibitor-treated cardiomyocytes perfused with 100 µM Diazoxide followed by 1 μM FCCP and 1 mM NaCN. For (A,B), N = 8 for vehicle-treated and N = 7 for ROMK inhibitor-treated. For (D,E), N = 19 cells for vehicle-treated, N = 21 cells for ROMK inhibitor; * p < 0.05. Comparisons in (B) were made with Student’s t-test. Two-way ANOVA was performed to assess differences during diazoxide perfusion.

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  Figure 5. Mitochondrial superoxide generation in isolated cardiomyocytes with and without ROMK inhibition. (A) Representative images and cumulative data of isolated adult murine cardiomyocytes stained with mitoSOX red and treated with vehicle or ROMK inhibitor (30 μM Compound A) before and after perfusion with 1 mM H2O2. (B) Absolute mitoSOX fluorescence of all cells at baseline prior to perfusion treated with vehicle or ROMK inhibition. (C) Representative images and cumulative data of isolated adult murine cardiomyocytes stained with mitoSOX red and treated with vehicle or ROMK inhibitor (30 μM Compound A) before and after perfusion with 4 μM Antimycin. (D) Representative images and cumulative data of isolated adult murine cardiomyocytes stained with mitoSOX red and treated with vehicle or ROMK inhibitor (30 μM Compound A) before and after perfusion with 10 μM Rotenone. For panels (A,C,D) pink cumulative line traces are ROMK inhibitor treated cells while gray lines represent vehicle treated cells. N = 4 mice; for H2O2, N = 11 cells for vehicle, N = 13 cells for ROMK inhibition; for Rotenone, N = 12 cells for vehicle, N = 19 cells for ROMK inhibition; for antimycin, N = 12 cells for…

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  Figure 6. The regulation of mitochondrial membrane potential (ψm) by ATP in isolated mouse heart mitochondria. (A) Representative traces of the effect of low (100 µM) or high (1000 µM) ATP on ψm in the presence or absence of 1 µg PIP2. (B) Dose–response curves of Δψm to different [ATP] with and without 1 µg of PIP2. (C) The effect of high doses of ATP on ψm in the presence or absence of 1 µg PIP2. N = 3–5 trials/group. * p < 0.05, ** p < 0.01.

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  Figure 7. The effect of pH on the regulation of mitochondrial membrane potential (ψm) by ATP in isolated mouse heart mitochondria. (A) Representative traces of the effect of 3000 µM ATP on ψm at a pH of 7.0 vs. 8.0. (B) Dose–response curve of Δψm treated with 3000 µM ATP at different pH levels. N = 3–5.

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  Figure 8. Exaggerated effect of ATP hyperpolarization on ψm after ROMK inhibition. (A) Representative traces of the effect of 1000 µM ATP on ψm with or without ROMK inhibition in the setting of PIP2. (B) Cumulative data of Δψm after 1000 µM ATP treatment with and without ROMK inhibition and PIP2. (C) Mitochondrial swelling measured by light scattering at 540 nm using a spectrophotomer normalized to swelling induced by mitochondria treated with FCCP. Conditions either had the presence of 1000 µM of ATP or the absence of ATP. The effect on mitochondrial swelling of different concentrations of the ROMK inhibitor, Compound A (0, 1, 3, 10 µM), was assessed. N = 4–6 trials/group for ψm experiments; N = 9 trials/group for swelling experiments; * p < 0.05. Comparisons between groups were made with Student’s t-test.

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  Figure 9. Proposed hypothesis for cardioprotection from inhibition of cardiac mitochondrial ROMK. Increased K+ in the mitochondrial matrix leads to several observed events that are associated with the prevention of ischemic injury. CCDC51 serves primarily as a mitochondrial K+ influx channel. The inherent properties of ROMK2 as an inward rectifying K+ channel and conditions during ischemia may promote ROMK2 to behave as a K+ efflux channel, which includes pH changes, increased matrix [K+], mitochondrial membrane potential depolarization, and activation of protein kinases. The blockade of ROMK2 during ischemia, we propose, increases mitochondrial matrix K+. We hypothesize that the inhibition of ROMK2 represents a novel therapy in ischemic heart disease.