Targeted SERCA2a gene expression identifies molecular mechanism and therapeutic target for arrhythmogenic cardiac alternans

Abstract

Background: Beat-to-beat alternans of cellular repolarization is closely linked to ventricular arrhythmias in humans. We hypothesized that sarcoplasmic reticulum calcium reuptake by SERCA2a plays a central role in the mechanism of cellular alternans and that increasing SERCA2a gene expression will retard the development of cellular alternans.

Methods and results: In vivo gene transfer of a recombinant adenoviral vector with the transgene for SERCA2a (Ad.SERCA2a) was performed in young guinea pigs. Isolated myocytes transduced with Ad.SERCA2a exhibited improved sarcoplasmic reticulum Ca(2+) reuptake (P<0.05) and were markedly resistant to cytosolic calcium alternans (P<0.05) under repetitive constant action potential clamp conditions (ie, when alternation of action potential duration was prevented), proving that sarcoplasmic reticulum Ca(2+) cycling is an important mechanism in the development of cellular alternans. Similarly, SERCA2a overexpression in the intact heart demonstrated significant resistance to alternation of action potential duration when compared with control hearts (heart rate threshold, 484+/-25 bpm versus 396+/-11 bpm, P<0.01), with no change in action potential duration restitution slope. Importantly, SERCA2a overexpression produced a 4-fold reduction in susceptibility to alternans-mediated ventricular arrhythmias (P<0.05).

Conclusions: These data provide new evidence that sarcoplasmic reticulum Ca(2+) reuptake directly modulates susceptibility to cellular alternans. Moreover, SERCA2a overexpression suppresses cellular alternans, interrupting an important pathway to cardiac fibrillation in the intact heart.

Figure 1

Gene transfer efficiency 72 hours after in vivo viral transduction. Panel A: Example of X-gal stained cross section of guinea pig ventricles excised 4 days after Ad.β-gal exposure. Panel B: Myocytes isolated from left ventricular free wall of guinea pig heart transduced with Ad.SERCA2.GFP. Fluorescent image shows the GFP transduction efficiency around 30%. Panel C: Example of protein expression. In vivo viral transduction increased SERCA2a expression compared to control (p<0.05).

Figure 2. Ca²⁺ cycling characteristics

Intracellular Ca transients and action potentials were induced under current clamp conditions at a pacing rate of 150 bpm at 30°C. Panel A: Traces were recorded from a control myocyte (left) and an Ad.SERCA2a myocyte (right). The Ca cycling characteristics in the Ad.SERCA2a myocyte were different from those in the control myocyte as illustrated by faster Ca reuptake (smaller Ca decay time constant (τ), shorter Ca transient duration), and greater Ca release (greater Ca amplitude). However, diastolic [Ca²⁺] and action potential durations were not statistically different between these cells. Panel B: Summary data from Ad.SERCA2a myocytes (n=6) and control myocytes (n=6) are shown. As compared to control myocytes, Ad.SERCA2a myocytes exhibited markedly faster Ca reuptake (38% faster τ) and greater Ca release (43% larger amplitude).

Figure 3. SERCA2a overexpression suppresses cellular alternans in isolated myocytes

Panel A: Representative examples of action potential and Ca transient recordings obtained from an Ad.SERCA2a myocyte and control myocyte. Traces recorded from two consecutive beats are superimposed to illustrate alternans. APD-ALT and Ca-ALT were induced as stimulation rate was increased to 200 bpm in the control myocyte, whereas alternans was not initiated until a pacing rate of 375 bpm in the Ad.SERCA2a myocyte. Panel B: SERCA2a overexpression increased alternans threshold and decreased alternans magnitude in isolated myocytes. Plot of pacing rate versus magnitude of APD-ALT from summary data of control myocytes (n=9) and Ad.SERCA2a myocytes (n=11) shows that the magnitude of APD-ALT increased as pacing rate increased, and the magnitude of APD-ALT was consistently greater in control myocytes compared with Ad.SERCA2a myocytes. The inset shows that under current clamp conditions, the threshold stimulation rate for both APD-ALT and Ca-ALT in control myocytes (n=9) is significantly lower than in Ad.SERCA2a myocytes (n=9) (265±17 bpm and 349±22 bpm respectively, p<0.01).

Figure 4. SERCA2a overexpression increased alternans threshold even under constant action potential clamp conditions

Panel A: Ca-ALT occurred under constant action potential clamp conditions. The top trace is action potential clamp protocol (voltage command). In this example, Ca transients recorded under constant action potential clamp conditions at stimulation rate of 200 bpm are shown in the middle (Ad.SERCA2a myocyte) and in the bottom (control myocyte). At this stimulation rate Ca transients alternate in the control myocyte but not in the Ad.SERCA2a myocyte. Panel B: The differences in threshold for Ca-ALT between control (n=12) and Ad.SERCA2a myocytes (n=4) remained even under constant action potential clamp conditions (254±11 bpm and 352±26 bpm respectively, p<0.01).

Figure 5. SERCA2a overexpression increased alternans threshold and decreased alternans magnitude in the whole heart

Plot of pacing rate versus magnitude of APD-ALT from Ad.SERCA2a, control Langendorff-perfused whole hearts shows that the magnitude of APD-ALT increased as pacing rate increased, and the magnitude of APD-ALT was consistently greater in control (n=8) hearts compared with Ad.SERCA2a (n=4) hearts. The inset shows that the threshold pacing rate for APD-ALT in control hearts is significantly lower than Ad.SERCA2a hearts (396±11 bpm and 484±25 bpm respectively, p<0.01).* p≤0.01 Ad.SERCA2a vs control.

Figure 6. SERCA2a overexpression does not change dynamic APD restitution in the whole heart

Plot of mean dynamic APD restitution shows no difference between control and Ad.SERCA2a hearts. The inset shows that the actual threshold pacing rate for alternans was lower in control vs. Ad.SERCA2a hearts yet, there was no significant difference in maximum APD restitution slope or the predicted alternans thresholds.

Figure 7. SERCA2a overexpression suppresses pacing-induced ventricular arrhythmias

Panel A: Representative optical action potential tracing from a control heart at a pacing CL of 140 ms demonstrating APD-ALT immediately preceding the onset of ventricular fibrillation. Conversely, in a Ad.SERCA2a heart at an even faster pacing CL(130 ms) there is little to no APD-ALT and the absence of an arrhythmia. Panel B: Using a ramp pacing protocol 8/8 control either VT or VF. In contrast, only 1 of 4 Ad.SERCA2a hearts developed a ventricular arrhythmia (p<0.05). * p<0.05 Ad.SERCA2a vs control.