Dual gene therapy with SERCA1 and Kir2.1 abbreviates excitation without suppressing contractility (original) (raw)
Ectopic expression of SERCA1a protein in the infected hearts. We first sought to verify the expression of SERCA1 protein after intramyocardial injection in the S-K group. Western blot analysis was performed using cardiac homogenates to detect SERCA1a protein expression. Figure 2 shows that a SERCA1a-specific antibody (14) detected a band (∼100 kDa) in the AdESERCA1-Kir2.1–injected guinea pig hearts, but not in AdEGI-injected animals, where only SERCA2 was evident. Calcium transients. Cardiac myocytes isolated from humans and from several animal models of heart failure typically exhibit intracellular Ca2+ transients with decreased amplitudes (13, 16) consistent with decreased contractility, which is the hallmark of heart failure. The SERCA2a in the heart normally maintains appropriate Ca2+ loading of the sarcoplasmic reticulum (SR) , thereby serving as a key modulator of releasable Ca2+ during excitation-contraction coupling. Heart failure is generally associated with decreased Ca2+ loading of the SR, so that supplementation of SERCA is a logical goal of therapy to reverse contractile dysfunction (17). Here, our primary goal was to preserve systolic function in the face of the abbreviation of excitation by coexpressed K channels.
SERCA protein expression. When an mAb against SERCA1a (A52; ref. 14) was assayed, a specific band at about 100 kDa was observed in samples from AdESERCA1-Kir2.1–injected hearts, while no signal was detected in control heart samples. Interestingly, the density of the band correlates well with the amount of virus injected (1.6× vs. 1×). When a specific antibody against SERCA2 was assayed, the right band appeared in all heart samples (AdESERCA1-Kir2.1–injected and control) with the same magnitude. GP, guinea pig.
To determine the functional consequences of SERCA1 ectopic expression in the absence of superimposed changes of excitability, we measured Ca2+ transients during depolarizing voltage steps in freshly isolated cardiomyocytes from the left ventricle of injected guinea pigs. The results, shown in Figure 3, revealed a significant increase in the amplitude of Indo-1 fluorescence ratios in the AdESERCA1-Kir2.1–infected cells compared with control myocytes (noninfected cells of the same hearts and AdEGI-infected myocytes) (1.56 ± 0.14, n = 6, vs. 1.00 ± 0.12, n = 8; P < 0.05, t test). Thus, SERCA1 overexpression in the bicistronic vector enhanced the availability of activator Ca2+, a functional effect which may be salutary in heart failure and which would tend to offset the effects of K channel overexpression. Note that these calcium transients were deliberately measured under voltage clamp, with identical voltage step durations and amplitudes in both groups, to define changes of calcium availability independent of any changes in APD or morphology.
Intracellular Ca2+ transients. Representative Ca2+ transients elicited in control (a) and AdESERCA1-Kir2.1–infected myocytes (b) are shown. AdESERCA1-Kir2.1 infection increased the amplitude of the Indo-1 fluorescence ratio. (c) Average data are summarized (n = 5 for AdESERCA1-Kir2.1 group, and n = 8 for control). *P < 0.05, control versus S-K group, t test.
Overexpression of Kir2.1 protein. We next sought to verify the overexpression of Kir2.1 in the S-K group. Here, we cannot take advantage of differentially specific antibodies, as Kir2.1 is normally expressed in the ventricle. Thus, we performed quantitative immunoblots using an antibody against Kir2.1 protein and compared the expression by densitometry to that of actin (which was assumed not to change). An increase of expression was detected in cardiac homogenates from AdESERCA1-Kir2.1–infected hearts (Figure 4). Nevertheless, given the uncertainties of interpreting relatively small percent changes in immunoblots, we next sought functional evidence of an increased density of inward rectifier K channels in cardiomyocytes from the S-K group.
Kir2.1 protein expression. (a) Western blot analysis using a polyclonal antibody against Kir2.1 protein. Although the specific band at approximately 42 kDa was present in both samples, its density was greater in the AdESERCA1-Kir2.1–injected guinea pig heart. (b) The relative expression of Kir2.1 normalized per actin protein for the same Western blot. Kir2.1 expression in the AdESERCA1-Kir2.1–injected heart was 30% higher than in control.
Overexpression of IK1 in guinea pig myocytes. As shown in Figure 5, 72 hours after injection and stimulation with GS-E, freshly isolated infected myocytes from the S-K group exhibit a significant increase in the inwardly rectifying IK1 density. The increase is particularly evident at negative membrane potentials where conductance is large (i.e., –54.8 ± 3.8 pA/pF vs. –30.7 ± 3.6 pA/pF at –140 mV, n = 8 for each group; P < 0.001, two-way ANOVA). We tested the functional importance of Kir2.1 overexpression at less negative potentials by examining the effect of AdESERCA1-Kir2.1 infection on AP repolarization.
Inward rectifier K current (IK1). AdESERCA1-Kir2.1–infected myocytes showed a significant increase in IK1 amplitude at negative membrane potentials when compared with control myocytes (AdEGI-infected or noninfected cells). *P < 0.01, two-way ANOVA; n ≥ 8 for each point.
APD. Prolongation of the AP is characteristic of ventricular myocytes isolated from animals and humans with heart failure (13, 18–21). Changes in the APD result from alterations in the functional expression of depolarizing and repolarizing currents that are active during the plateau phase. Repolarization in the mammalian heart is achieved primarily by the activity of potassium-selective ionic currents. IK1 contributes to the terminal phase of repolarization, so an increase in the expression of these channels is a logical mechanism to shorten the APD. Figure 6 shows steady-state APs stimulated at 1 Hz in freshly isolated control and S-K ventricular myocytes at 37°C in the absence of added calcium buffer to the cytosol. The SERCA-Kir2.1–infected myocyte APs were significantly shorter than control myocyte APs, at both 50% and 90% repolarization (188.8 ± 35.9 ms vs. 292.2 ± 15.6 ms [APD50] and 220.1 ± 38.8 ms vs. 346.7 ± 18.6 ms [APD90], for S-K myocytes [n = 8] vs. for control cells [n = 13], respectively; P < 0.05, t test).
APD. (a and b) Representative action potentials elicited in control and AdESERCA1-Kir2.1–infected myocytes. AdESERCA1-Kir2.1 infection abbreviated the AP. (c) Average APD50 and APD90 data are summarized (n = 8 for AdESERCA1-Kir2.1 group, and n = 13 for control). *P < 0.05, AdESERCA1-Kir2.1 versus control, t test.
We acknowledge that the electrophysiologic and Ca2+ handling pathways in the heart interact and, as such, may have impacted the degree of AP abbreviation achieved via genetic manipulation of SERCA1 and Kir2.1 channels. We tested whether SERCA1 overexpression, by altering intracellular Ca2+ handling, had potentially increased or decreased the effect of Kir2.1 overexpression on abbreviation of APD via Ca2+-dependent modulation of membrane potential. Cellular AP recordings were made under identical conditions, with one exception: the addition of the cytosolic Ca2+ buffer EGTA (5 mM). By buffering the Ca2+ transient, we eliminated Ca2+-dependent modulation of membrane potential exerted by the Ca2+-sensing actions of the L-type Ca current, the electrogenic Na+/Ca2+ exchange, and other Ca2+ actions on currents. Under these Ca2+-buffered conditions, APs in control cells (APD50 = 302.7 ± 82.8 ms, APD90 = 376.8 ± 79.8 ms, n = 6) were slightly longer and APs in S-K cells (APD50 = 147.9 ± 28.9 ms, APD90 = 194.8 ± 22.5 ms, n = 5) were shorter on average compared with APs recorded in the absence of any added Ca2+ buffer (not significant). Thus, via this experimental assessment, coexpression of SERCA1 moderated the impact of Kir2.1-overexpressed channels to shorten the AP.
These data demonstrate the intended therapeutic effect of overexpression of Kir2.1 at the cellular level. As long as SERCA-Kir2.1 overexpression does not aggravate contractile dysfunction, increased IK1 current would be expected to reduce the incidence of ventricular arrhythmias in failing hearts, by stabilizing repolarization and by suppressing triggered activity.
Widespread ectopic expression of SERCA1a protein. Prior to performing cardiac function measurements, we verified widespread expression of SERCA1a protein throughout the left ventricle of animals that underwent multiple-site injections of AdESERCA1-Kir2.1. Western blot analysis was performed using homogenates of right and left ventricle and the SERCA1a-specific mAb A52 (14). The right ventricle was sectioned into two samples (Figure 7, lanes 2 and 3) and the left ventricle was cut into six similarly sized pieces (lanes 4–9). Figure 7 shows the Western blots from two different animals 72 hours after injection. In both blots, the first lane corresponds to a homogenate of guinea pig skeletal muscle used as a positive control for the antibody. Detection of the approximately 100-kDa SERCA1a-specific band in the immunoblots documents that widespread expression of ectopic SERCA1a protein was obtained throughout the entire left ventricle (lanes 4–9) in one animal and in five of six sections (lanes 4–8) of left ventricle in the other animal in which Western blot analysis was performed to assess the extent of protein expression. In contrast, no SERCA1a expression was observed in the noninjected right ventricle (lanes 2 and 3).
Widespread SERCA1 expression. Western blots from two different animals subjected to multiple-site injections of AdESERCA1-Kir2.1 throughout the left ventricle are shown. In both blots, the first lane corresponds to a homogenate of guinea pig skeletal muscle as a positive control for the SERCA1a-specific mAb A52 (14). Lanes 2 and 3 are the samples of the right ventricle, and lanes 4–9 correspond to each of six pieces into which the left ventricle was cut. Widespread expression of ectopic SERCA1a protein (∼100-kDa band) was obtained in the injected left ventricle, whereas no SERCA1a expression was observed in the noninjected right ventricle.
ECG recordings. Arrhythmias are a major cause of death in heart failure (22, 23). Cardiomyocytes from failing hearts reveal abnormalities in repolarization with prolongation of the QT interval, basically due to downregulation of K+ currents, that favor the development of such arrhythmias. ECG recordings were performed in animals that had undergone widespread injection of the adenovirus vectors into the left ventricular myocardium. The QT interval was measured and corrected for heart rate using the following formula, with all measurements in seconds: QTc = QT / √cyclelength.
As shown in Figure 8, the QTc interval measured at 72 hours after injection and stimulation with GS-E was significantly abbreviated in the AdESERCA1-Kir2.1–injected guinea pigs compared with measurements made in the same animal immediately after surgery (0.26 ± 0.02 s vs. 0.36 ± 0.02 s; P < 0.03, paired t test). In contrast, no changes in the QTc interval were observed in the animals injected with AdEGI (0.33 ± 0.01 s vs. 0.33 ± 0.03 s; not significant). There was no difference, either, in the QTc interval recorded immediately after surgery between the two groups of animals (AdESERCA1-Kir2.1– and AdEGI-injected; data not shown).
ECG analysis: QTc interval. A significant reduction was observed in the QTc interval in AdESERCA1-Kir2.1–injected animals (black bars). No changes occurred in the animals treated with AdEGI. *P = 0.003, immediately after surgery versus 72 hours after injection and stimulation with GS-E, paired t test.
Cardiac echocardiography. Table 1 displays the data from the cardiac echocardiography measurements. There were no significant differences in the left ventricular dimensions in the AdEGI versus the AdESERCA1-Kir2.1 animals. Left ventricular function as assessed by shortening fraction was also highly similar in the two groups. Heart rates were slightly higher, though not significantly so in the AdESERCA1-Kir2.1 animals. The septal and left ventricle free wall thicknesses were also not significantly different between the two groups (data not shown), indicating no pathologic hypertrophy developed in the animals. Although the AdESERCA1-Kir2.1 animals had significantly shortened APD and QT intervals, the function was not impaired, perhaps due to increased SR pumping efficiency related to the SERCA1 overexpression.
Echocardiography measurements in guinea pigs 72 hours after viral injection