Project 12: Role of spontaneous cellular Calcium release events in the development of ectopic activity in atrial tissue from patients with atrial fibrillation

PI Göttingen: N. Voigt; PI London: Q. Zhang; PhD student: Julius R.D. Pronto

Scientific background and preliminary results

Atrial fibrillation (AF) is the most common sustained arrhythmia in the clinical setting and is associated with significant morbidity and mortality. Currently available AF therapies have limited efficacy and safety, particularly in patients with long-standing persistent (‘chronic’) AF (cAF).1 A better understanding of the molecular mechanisms promoting AF is expected to facilitate the development of improved anti-AF therapies. In general, ectopic (triggered) activity and reentry have been identified as the most prominent arrhythmogenic mechanisms.1 We and others have shown that AF is associated with electrical remodeling of various ion currents, resulting in reentry-promoting shortening of atrial repolarization.1 Increased open probability of the sarcoplasmic reticulum (SR) Ca2+-release channel (ryanodine receptor channel type-2, RyR2) predisposes patients with paroxysmal (pAF) and cAF to spontaneous (non-AP-triggered) diastolic SR Ca2+ release events (SCaEs).2-5 Resulting excess diastolic [Ca2+]i is handled by Na+/Ca2+ exchanger (NCX), which by transporting three Na+ ions into the cell per single Ca2+ ion extruded, creates a depolarizing transient inward current (“Iti”) that can produce arrhythmogenic membrane depolarizations (delayed afterdepolarizations, DADs).

However, the exact role of SCaEs and cellular DADs in AF initiation and maintenance remains controversial.6 Although SCaEs and cellular DADs occur more often in myocytes from patients with pAF and cAF, depolarization of a single myocyte within healthy cardiac tissue is not expected to cause appreciable depolarization, since surrounding cells, coupled by gap-junctions, keep the membrane potential too negative for initiation of a new AP.7 Accordingly, human atrial trabeculae preparations from cAF patients were shown to produce less abnormal aftercontractions compared to sinus rhythm (Ctl) patients.8

On the other hand, previous studies in human atrial trabeculae revealed impaired contractile response of atrial trabeculae from cAF patients.9 Our preliminary experiments point to impaired contractile response of atrial myocytes from patients with cAF to electrical excitation. Atrial cardiomyocytes from patients with cAF were ~30% longer compared to Ctl and the fractional shortening (ΔL/L0) of the myocytes in the cAF group was impaired. Therefore we hypothesize that impaired cellular contractility masks the occurrence of spontaneous aftercontractions in atrial trabeculae from patients with cAF despite the increased incidence of SCaE and cellular DADs that are likely to trigger more atrial ectopic excitations in atrial tissue preparations from cAF patients.

Hypotheses of the PhD project

In the proposed project we will test the hypotheses that

(i) SCaEs and related DADs occur in myocytes in intact tissue preparations obtained from patients with pAF and cAF.

(ii) Local cellular SCaEs and DADs can trigger electrical excitations travelling through the atrial tissue preparation in patients with pAF and cAF.

(iii) Impaired cell-cell coupling promotes the occurrence of cellular DADs and triggered APs in patients with pAF and cAF.

Work programme

Fresh slices (300 µm) from right atrial appendages from patients undergoing open heart surgery will be prepared using a vibratome. Slices will be incubated at room temperature with the Ca2+ indicator Fluo-8 AM (10 µM for 20 min). Contraction will be inhibited by adding blebbistatin (2 µM) to the bath solution.

Slices will be stimulated using a point electrode at 1 Hz and 6 Hz (20% above threshold). Imaging will be performed with an inverted microscope and a fast imaging camera (ANDOR iXonEMplus). Analysis of diastolic and systolic [Ca2+]i values, amplitude and decay of CaT will provide first insights into alterations of Ca2+ homeostasis. Since increased incidence of SCaEs contributes to cellular pathophysiology of pAF and cAF patients,2-5 foci of SCaEs will be identified and Ca2+ wave propagation to neighboring cells will be analyzed. In case SCaEs cannot be elicited, we will increase Ca2+ concentration in the bath solution to 5 mM to challenge the system and enhance the susceptibility to SCaEs.4,10

We will load the slices with the voltage sensitive dye RH-237 (10 µM, acute applicaton). Action potentials will be recorded optically during point stimulation at 1 Hz and 6 Hz. Propagation velocity will be analyzed in slices from Ctl, pAF and cAF patients. To test whether cellular SCaEs are sufficient to generate DADs in intact tissue preparations, we will stop stimulation and record membrane potential during one minute follow up. We will investigate whether cellular DADs are sufficient to depolarize neighboring cells and generate a new AP (ectopic activity).

It has been suggested that gap junction coupling between neighboring myocytes stabilizes myocytes and prevents occurrence of DADs initiated by SCaEs.7 In order to test this hypothesis, we will enhance gap junctional coupling between myocytes pharmacologically using rotigaptide (50 nM). Conversely, we will employ carbenoxolone (25 µM) to suppress gap junctional cardiomyocyte interaction and limit the current sink ordinarily created by well coupled myocyrdium. In order to investigate gap junctional coupling directly, we will load slices with calcein (10 µM, 20 min). Calcein diffusion between cells will be employed as marker for cell-cell coupling and quantified as calcein fluorescence recovery after photobleacing.7

In order to further investigate molecular basis for alterations in coupling between atrial cardiomyocytes we will image expression and distribution major atrial gap-junctional proteins connexin 40 and connexin 43 (Cx40 and Cx43) in right atrial samples from Ctl, pAF and cAF patients. Since altered Cx phosphorylation has been suggested to underlie lateralization of Cx in the atria from animal models,11 we will investigate phosphorylation of Cx40 and Cx43 using phosphospecific antibodies.

Finally, mitochondrial oxidative stress has been suggested to contribute to Cx remodeling in patients with heart failure.12 In order to test whether similar mechanisms may be involved in cAF associated Cx remodeling, we will incubate atrial tissue slices with MitoTEMPO (1 µM, 1 hour), a membrane-permeable ROS scavenger targeted to mitochondria. We will investigate Cx expression and phosphorylation in those tissue slices and occurrence of SCaEs and DADs as described above.

Taken together, the suggested experiments will reveal important insights into the role of cellular SCaE and DADs in the pathophysiology of atrial arrhythmias.

Prof. Dr. med. Niels Voigt
Molecular Pharmacology
+49 (0)551-39 22602

Research interests: Atrial fibrillation, cardiomyocyte Ca2+ signalling

Dr. Qiuping Zhang
Cardiovascular Division, KCL

Research interests: Nesprin, nuclear envelope LINC complex, cardiomyocyte mechanotransduction and cardiomyopathies

Julius R. D. Pronto
PhD student 3rd cohort

RP 12.3: Role of spontaneous cellular Calcium release events in the development of ectopic activity in atrial tissue from patients with atrial fibrillation