Evidence Against the Role of Intracellular Calcium Dynamics in Ventricular Fibrillation
To the Editor:
In our recent publication,1 we argued that intracellular calcium (Cai) dynamics is not an essential mechanism of the maintenance of ventricular fibrillation (VF). Our argument was based on 2 major observations: (1) Cai transient passively followed the optical action potential along the entire wavelet span, except at the very wavelet tip; and (2) chelating Cai with BAPTA-AM did not change the incidence of wave break (WB) during VF. In their letter to Circulation Research, Ogawa et al2 raised a concern that our BAPTA test is not valid because the time of BAPTA perfusion in our experiments (≈10 minutes) was too short to achieve a significant effect. To support this statement, Ogawa et al referred to their own (unpublished) results in Langendorff-perfused rabbit hearts indicating that after 30 minutes of perfusion with 20 μmol/L BAPTA-AM, rapid pacing caused Cai transient alternans and eventual initiation of VF, whereas, after 70 minutes of BAPTA-AM perfusion, Cai transient alternans was no longer present and only monomorphic ventricular tachycardia could be induced.2
In response to this critique, we first mention that duration of BAPTA-AM infusion necessary to achieve suppression of Cai transient may vary depending on experimental conditions. The observation reported by Ogawa et al2 that 30-minute perfusion with BAPTA-AM was not sufficient to achieve the effect cannot be extended to the general case. For example, Marbán et al3 were able to achieve complete abolishment of Cai transient and contraction after 10 to 20 minutes of BAPTA-AM infusion. Thus, the duration of BAPTA-AM infusion is hardly an adequate or sufficient parameter to assess the drug effect. Some estimation of Cai transient amplitude and/or contractility should be provided to ensure that the effect is indeed present. Unlike the unpublished results referred to by Ogawa et al,2 our published results1 included direct assessment of BAPTA-AM effects. Specifically, we showed that the left ventricular developed pressure (LV-devP) and Cai transient amplitude were significantly decreased after BAPTA-AM infusion (to 12.2% and 30.5% of control values, respectively). Note that nonlinearity of Rhod-2 fluorescence with respect to calcium concentration should overestimate the amplitude of residual Cai transient after BAPTA-AM infusion. Because it was not feasible or at least practical to calibrate Rhod-2 signal in our study, we relied more on LV-devP as an indicator of BAPTA-AM effectiveness. Because, despite 88% decrease in LV-devP, WB incidence remained unchanged,1 we reasoned that Cai cycling is not a major mechanism of VF maintenance.1
One may argue that even this residual level of Cai cycling can still be sufficient to provide the hypothetical destabilizing feedback between the Cai transient and the action potential leading to WB.4,5 To make this possibility even less likely, we performed additional experiments focusing on the goal of complete abolishment of Cai transient with BAPTA-AM. It was not an easy task. We could not follow the protocol described by Ogawa et al2 because, in our experience, long (>20 minutes) perfusion of porcine hearts with crystalline solutions results in edema, which in itself may lead to slowing and regularization of VF.6 On the other hand, when BAPTA-AM is infused into the blood circulation, a significant amount of the drug is absorbed by blood cells, preventing efficient delivery to the cardiomyocytes. After some experimentation aimed at enhancing BAPTA-AM delivery, while keeping edema to a minimum, we settled on the following protocol (N=2): after the initial 10-minute BAPTA-AM infusion,1 we performed 5 additional short (2- to 3-minute) infusions of BAPTA-AM dissolved in Tyrode’s solution separated by 9- to 10-minute perfusion with blood. This was followed by a final 10-minute BAPTA-AM infusion identical to the first infusion.1 The entire infusion protocol took ≈70 minutes. At the end of this protocol, LV-devP dropped to <5% of control, and Cai transients were virtually undetectable. Still, VF was inducible with rapid pacing, and the movies of the action potential showed multiple WBs. Thus, we affirm our position1 that, in the blood-perfused porcine heart, Cai dynamics is not necessary for VF maintenance. In addition, our new data indicate that Cai dynamics is not necessary for VF induction by rapid pacing.
Sources of Funding
Supported by NIH grant 1R01HL088444-01A1 and a research grant from the Nora Eccles Treadwell Foundation.
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