Abstract 322 Decreased cardiac troponin C underlies the altered intracellular Ca2+ buffering observed in atrial myocytes of patients with long-standing persistent atrial fibrillation

Background:
Long-standing persistent atrial fibrillation (AF) is associated with impaired atrial Ca²⁺ signalling and diminished levels of myofilament proteins which contribute to atrial hypocontractility and thromboembolic events. Although the myofilament protein, cardiac troponin C (cTnC), is known to buffer intracellular Ca²⁺, its contribution to the Ca handling abnormalities associated with AF is still poorly investigated. Therefore, this study aimed to elucidate the role of cTnC in AF-associated Ca²⁺ handling alterations.

Methods:
Membrane currents (patch-clamp technique) and cytosolic Ca²⁺ (Fluo-3 AM and Fluo-4 AM) were measured in right atrial myocytes from sinus rhythm (Ctrl) and AF patients, as well as atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with knocked down cTnC levels (si-iPSC-CM). Immunoblotting was employed to determine protein levels of myofilament proteins.

Results:
Protein levels of the cardiac troponins cTnC and cTnI were reduced in AF atrial tissues compared to Ctrl. Cellularly, both groups had similar SR Ca²⁺ content, which was calculated by integrating Na⁺-Ca²⁺ exchange current (I_NCX) during caffeine-induced release (AF: 1.62±0.53pC/pF, n/N=19/13; Ctrl: 1.79±0.89pC/pF), n/N=23/19; P=0.703). However, intracellular calcium buffering, estimated by fitting a hyperbolic function on buffering curves generated during the application of caffeine to obtain buffering parameters (Maximum buffer capacity, Bmax and dissociation constant of buffers, Kd), was impaired in AF, which could be due to the reduced cardiac troponins (Figure A and B). Interestingly, the reduction of cTnC levels in iPSC-CM (si-iPSC-CM) recapitulated the impaired buffering observed in AF myocytes (Figure C). Ca²⁺ sparks measurements revealed a higher Ca²⁺ spark frequency in si-iPSC-CM compared to the Ctrl group (Figure D).

Conclusions:
Reduced cTnC underpins the impaired intracellular Ca²⁺ buffering observed in AF, which could contribute to the arrhythmogenic substrate by promoting spontaneous SR Ca²⁺ release events that could trigger delayed afterdepolarizations.

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02087-y
Decreased cardiac troponin C underlies the altered intracellular Ca2+ buffering observed in atrial myocytes of patients with long-standing persistent atrial fibrillation
D. Hubricht¹, F. E. Fakuade², I. Sobitov², N. Ignatyeva¹, A. Ebert¹, H. Baraki³, I. Kutschka³, N. Voigt²
¹Herzzentrum, Klinik für Kardiologie und Pneumologie, Universitätsmedizin Göttingen, Göttingen; ²Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; ³Klinik für Herz-, Thorax- und Gefäßchirurgie (HTG), Universitätsmedizin Göttingen, Göttingen;
Background: Long-standing persistent atrial fibrillation (AF) is associated with impaired atrial Ca²⁺ signalling and diminished levels of myofilament proteins which contribute to atrial hypocontractility and thromboembolic events. Although the myofilament protein, cardiac troponin C (cTnC), is known to buffer intracellular Ca²⁺, its contribution to the Ca handling abnormalities associated with AF is still poorly investigated. Therefore, this study aimed to elucidate the role of cTnC in AF-associated Ca²⁺ handling alterations. Methods: Membrane currents (patch-clamp technique) and cytosolic Ca²⁺ (Fluo-3 AM and Fluo-4 AM) were measured in right atrial myocytes from sinus rhythm (Ctrl) and AF patients, as well as atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with knocked down cTnC levels (si-iPSC-CM). Immunoblotting was employed to determine protein levels of myofilament proteins. Results: Protein levels of the cardiac troponins cTnC and cTnI were reduced in AF atrial tissues compared to Ctrl. Cellularly, both groups had similar SR Ca²⁺ content, which was calculated by integrating Na⁺-Ca²⁺ exchange current (I_NCX) during caffeine-induced release (AF: 1.62±0.53pC/pF, n/N=19/13; Ctrl: 1.79±0.89pC/pF), n/N=23/19; P=0.703). However, intracellular calcium buffering, estimated by fitting a hyperbolic function on buffering curves generated during the application of caffeine to obtain buffering parameters (Maximum buffer capacity, Bmax and dissociation constant of buffers, Kd), was impaired in AF, which could be due to the reduced cardiac troponins (Figure A and B). Interestingly, the reduction of cTnC levels in iPSC-CM (si-iPSC-CM) recapitulated the impaired buffering observed in AF myocytes (Figure C). Ca²⁺ sparks measurements revealed a higher Ca²⁺ spark frequency in si-iPSC-CM compared to the Ctrl group (Figure D). Conclusions: Reduced cTnC underpins the impaired intracellular Ca²⁺ buffering observed in AF, which could contribute to the arrhythmogenic substrate by promoting spontaneous SR Ca²⁺ release events that could trigger delayed afterdepolarizations.
https://dgk.org/kongress_programme/ht2022/aBS665.html