Distinct cellular and molecular mechanisms underlie functional remodeling of repolarizing K+ currents with left ventricular hypertrophy

Abstract

Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk, although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and inwardly rectifying (Kir) K(+) channel remodeling was examined in a mouse model of pressure overload-induced LVH, produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV myocytes and QT(c) intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (C(m)) was higher, and I(to,f), I(K,slow), I(ss), and I(K1) densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of the reduced current densities is the increase in C(m), I(K,slow) amplitudes were decreased and I(ss) amplitudes were increased in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and increased I(ss) amplitudes were more pronounced in TAC endocardial LV cells, whereas I(K,slow) amplitudes were selectively reduced in TAC epicardial LV cells. Consistent with the similarities in I(to,f) and I(K1) amplitudes, Kv4.2, Kv4.3, and KChIP2 (I(to,f)), as well as Kir2.1 and Kir2.2 (I(K1)), transcript and protein expression levels were similar in TAC and sham LV. Unexpectedly, expression of I(K,slow) channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was increased in TAC LV. Functional changes in repolarizing K(+) currents with LVH, therefore, result from distinct cellular (cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms.

Figure 1. Detection of LVH 7 days after TAC

A, Representative echocardiographic M-mode images of sham and TAC LV. Scale bars are 1 mm and 100 ms. AW and PW designate anterior and posterior wall thicknesses, respectively. B, LVM/BW in sham (n=18) and TAC (n=39) mice; individual values and means±SEM are plotted. *P<0.001. C, Mean±SEM mRNA expression levels (arbitrary units) of the hypertrophy markers ANF and β-MHC are significantly (*P<0.001; §P<0.05) higher in TAC (n=6) than in sham (n=6) LV.

Figure 2. Alterations in repolarizing K⁺ currents in TAC LV myocytes

Representative whole-cell Kv currents (A), evoked during 4.5-second voltage steps to potentials between −40 and +40 mV from a holding potential (HP) of −70 mV, and Kir currents (B), evoked during 350-ms voltage steps to potentials between −40 and −120 mV (HP=−70 mV) in sham and TAC LV cells. Mean±SEM I_peak (C), I_to,f (D), I_K,slow (E), I_ss (F), and I_K1 (G) densities in sham and TAC LV myocytes are plotted as a function of test potential. Relative changes in C_m (H), current amplitudes (I), and densities (J) in TAC compared with sham LV myocytes. Values in TAC and sham LV myocytes are significantly (*P<0.001, #P<0.01) different.

Figure 3. Action potential and surface ECG abnormalities with TAC

A, Mean±SEM resting membrane potentials (V_m), action potential amplitudes (APA), and durations at 50% (APD₅₀) and 90% (APD₉₀) repolarization in sham and TAC RV and LV myocytes. APD₉₀ values are significantly (#P<0.01) longer in TAC LV cells (supplemental Table III). B, Representative lead II ECGs from anesthetized sham and TAC mice before and after surgery. QT_c interval durations (C) and J wave amplitudes (D) in sham (n=8) and TAC (n=8) mice before and after surgery; individual values and means±SEM are plotted (§P<0.05).

Figure 4. Regional (EPI/ENDO) differences in the effects of LVH on repolarizing K⁺ currents and C_m

A, Representative Kv currents recorded, as described in the legend to Figure 2, from sham and TAC EPI and ENDO LV myocytes. B, C_m in sham and TAC EPI and ENDO cells. Mean±SEM I_peak (C), I_to,f (D), I_K,slow (E), and I_ss (F) densities in sham and TAC EPI and ENDO LV myocytes are plotted as a function of test potential. Relative changes in current amplitudes (G and H) and densities (I and J) in TAC, compared with sham, EPI (G and I) and ENDO (H and J) LV myocytes. Indicated values in TAC and sham myocytes are significantly (*P<0.001, #P<0.01, §P<0.05) different.

Figure 5. Transcript expression profiling in TAC and sham LV

A, Total RNA content in sham (n=6) and TAC (n=8) LV; individual values and means±SEM are plotted (#P<0.01). Mean±SEM RNA expression levels (arbitrary units) of control (B) and K⁺ channel subunit and regulatory (C) genes in sham and TAC LV and of K⁺ channel subunit and regulatory genes in sham and TAC EPI and ENDO LV (D) (n=6 mice in each group). Indicated values are significantly (*P<0.001, #P<0.01, §P<0.05) different in TAC and sham LV and in EPI versus ENDO (†P<0.001) samples.

Figure 6. Protein expression in TAC and sham LV

A, Total protein content in sham (n=3) and TAC (n=4) LV; individual values and means±SEM are plotted (#P<0.01). Representative Western blots of total protein expression of K⁺ channel subunits, as well as of the control proteins, transferrin receptor (TransR), β-actin, and filamin C, in sham and TAC LV (B), EPI (D), and ENDO (E). Quantification of relative differences in total protein expression in sham and TAC total LV (C) and in sham and TAC EPI and ENDO LV (F). Data are means±SEM (n=3 to 6), and indicated values are significantly (§P<0.05) different in TAC and sham LV or TAC and sham EPI or ENDO LV. As in wild-type LV, Kv4.2 expression is higher (†P<0.01) in (sham and TAC) EPI than ENDO LV.

Figure 7. Cell surface protein expression in TAC and sham LV

A, Representative Western blots of cell surface expression of K⁺ channel subunit and the control (TransR) proteins in sham and TAC LV. B, Quantification of relative differences in cell surface protein expression in sham and TAC LV. Data are means±SEM (n=3 to 6), and expression levels are significantly (#P<0.01, §P<0.05) different in TAC and sham LV.