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. 2009 Sep;20(9):1048-54.
doi: 10.1111/j.1540-8167.2009.01475.x.

Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity

Yau-Chi Chan  1 , Chung-Wah Siu, Yee-Man Lau, Chu-Pak Lau, Ronald A Li, Hung-Fat Tse
Affiliations

Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity

Yau-Chi Chan et al. J Cardiovasc Electrophysiol. 2009 Sep.

Abstract

Introduction: Normal heart rhythms originate in the sinoatrial node. HCN-encoded funny current (I(f)) and the Kir2-encoded inward rectifier (I(K1)) counteract each other by respectively oscillating and stabilizing the negative resting membrane potential, and controlling action potential firing. Therefore, I(K1) suppression and I(f) overexpression have been independently exploited to convert cardiomyocytes (CMs) into AP-firing bioartificial pacemakers. Although the 2 strategies have been largely assumed synergistic, their complementarity has not been investigated.

Methods and results: We explored the interrelationships of automaticity, I(f) and I(K1) by transducing single left ventricular (LV) CMs isolated from guinea pig hearts with the recombinant adenoviruses Ad-CMV-GFP-IRES-HCN1-AAA and/or Ad-CGI-Kir2.1 to mediate their current densities via a whole-cell patch clamp technique at 37 degrees C. Results showed that Ad-CGI-HCN1-AAA but not Ad-CGI-Kir2.1 transduction induced automaticity (181.1 +/- 13.1 bpm). Interestingly, Ad-CGI-HCN1-AAA/Ad-CGI-Kir2.1 cotransduction significantly promoted the induced firing frequency (320.0 +/- 15.8 bpm; P < 0.05). Correlation analysis revealed that the firing frequency, phase-4 slope and APD(90) of AP-firing LV CMs were correlated with I(f) (R(2) > 0.7) only when -2 >I(K1) >-4 pA/pF but not with I(K1) over the entire I(f) ranges examined (0.02 < R(2) < 0.4). Unlike I(f), I(K1) displayed correlation with neither the phase-4 slope (R(2)= 0.02) nor phase-4 length (R(2)= 0.04) when -2 > I(f) > -4 pA/pF. As anticipated, however, APD(90) was correlated with I(K1) (R(2)= 0.4).

Conclusion: We conclude that an optimal level of I(K1) maintains a voltage range for I(f) to operate most effectively during a dynamic cardiac cycle.

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Figures

Figure 1
Figure 1
Representative action potential waveforms and averaged steady-state I-V relationships of control (A, E), Ad-CGI-Kir2.1- (B, F), Ad-CGI-HCN1ΔΔΔ- (C, G), as well as Ad-CGI-HCN1ΔΔΔ/Ad-CGI-Kir2.1 (D, H)-tranduced LV CMs.
Figure 2
Figure 2
Bar charts showing (A) RMP or MDP; (B) APD90; (C) beating rate; and (D) phase 4 slope of control, Ad-CGI-Kir2.1-transduced, Ad-CGI-HCN1ΔΔΔ-transduced, as well as Ad-CGI-HCN1ΔΔΔ/Ad-CGI-Kir2.1-cotranduced LV CMs. *, P<0.05.
Figure 3
Figure 3
The effect of external application of Ba2+ on the automaticity in Ad-CGI-HCN1ΔΔΔ/Ad-CGI-Kir2.1-cotranduced LV CMs. Representative action potential waveforms at baseline, with 0.1 mM BaCl2, and 0.3 mM BaCl2 are shown, respectively.
Figure 4
Figure 4
(A) Correlations between If and the frequency of spontaneously AP-firing cells when IK1, -60mV ≥-2 pA/pF (■), -2>IK1, -60mV >-4 pA/pF (○) and IK1, -60mV ≤-4 pA/pF (▲). (B) Correlations between IK1 and the firing frequency rate when If, -60mV ≥-2 pA/pF (■),-2>If, -60mV >-4 pA/pF (○) and If, -60mV ≤-4 pA/pF (▲).
Figure 5
Figure 5
(A) Correlations between If and the phase 4 slope of spontaneously AP-firing cells when IK1, -60mV ≥-2 pA/pF (■), -2>IK1, -60mV >-4 pA/pF (○) and IK1, -60mV ≤-4 pA/pF (▲). (B) Correlations between IK1 and the phase 4 slope when If, -60mV ≥-2 pA/pF (■), -2>If, -60mV >-4 pA/pF (○) and If, -60mV ≤-4 pA/pF (▲). (C) Correlations between If and the phase 4 length of spontaneously AP-firing cells when IK1, -60mV ≥-2 pA/pF (■), -2>IK1, -60mV >-4 pA/pF (○) and IK1, -60mV ≤-4 pA/pF (▲). (D) Correlations between IK1 and the phase 4 length when If, -60mV ≥-2 pA/pF (■), -2>If, -60mV >-4 pA/pF (○) and If, -60mV ≤-4 pA/pF (▲). (E) Correlations between If and the APD90 of spontaneously AP-firing cells when IK1, -60mV ≥-2 pA/pF (■), -2>IK1, -60mV >-4 pA/pF (○) and IK1, -60mV ≤-4 pA/pF (▲). (F) Correlations between IK1 and the APD90 when If, -60mV ≥-2 pA/pF (■), -2>If, -60mV >-4 pA/pF (○) and If, -60mV ≤-4 pA/pF (▲).
Figure 6
Figure 6
Effects of adrenergic stimulation by superfusion of 1 μM isoproterenol for 5 minutes (3 cells from a single animal) on HCN1-ΔΔΔ-transduced LV CMs.
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