The alteration of calcium wave characteristics by K201 in rabbit cardiomyocytes provides an insight into the cellular mechanism underlying the anti-arrhythmic effect of the drug

ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015

I Ca inactivation was not significantly altered by sorcin following SR inhibition with thapsigargin (25 AM) (thapsigargin: s, 22.3 T 1.1, n = 15; thapsigargin + sorcin: s, 22.8 T 3.6, n = 7, < 0.05), suggesting that SR flux may be important in mediating the effects of sorcin. Substitution of Ca2+ for Ba2+ in the superfusate significantly slowed I Ca(Ba) inactivation to 75 T 11 ms (n = 11, P < 0.05). In the presence of sorcin, I Ca(Ba) inactivation was significantly reduced (s, 46.3 T 6 ms, n = 10, P < 0.05), suggesting that sorcin also mediates effects directly on LTCC. These data suggest that sorcin modulates LTCC inactivation by its inhibitory effect on RyR during normal LTCC and RyR coupling; uncoupling of LTCC and RyR unmasks a direct effect of sorcin on LTCC. Under normal conditions, the effect of sorcin on RyR dominates over the effect on I Ca. doi:10.1016/j.yjmcc.2006.03.033

019. The alteration of calcium wave characteristics by K201 in rabbit cardiomyocytes provides an insight into the cellular mechanism underlying the anti-arrhythmic effect of the drug Christopher Loughrey, Godfrey Smith. University of Glasgow, Glasgow, UK Enhanced diastolic Ca2+ release mediated by the cardiac ryanodine receptor (RyR2) is associated with an increased incidence of arrhythmias. The benzothiazepine derivative K201 developed by Kaneko et al., Drug Dev Res (1996) has been shown to reduce diastolic Ca2+ leak from SR vesicles via RyR2. The current study is the first to report the effects of K201 on Ca2+ waves in cardiomyocytes. These events are a form of diastolic Ca2+ release that has been linked to triggered arrhythmias. Confocal microscopy was used to visualise Ca2+ wave characteristics in permeabilised ventricular cardiomyocytes perfused with a mock intracellular solution containing 10 AM Fluo-3 (free acid), 350 nM [Ca2+]. Addition of 1 AM K201 for 90s significantly decreased Ca2+ wave frequency to 77 T 3.9%, (n = 6; P < 0.05) of control value while 3 AM (n = 7) completely abolished Ca2+ waves. 1 AM K201 significantly reduced mean Ca2+ wave velocity to 87 T 4.2% (P < 0.05) of control while velocity (of last wave) was reduced to 67.7 T 6.5% (P < 0.05) in 3 AM K201. No change in SR Ca2+ content was observed at 1 AM while 3 AM K201 reduced SR Ca2+ content to 73.4% of control (n = 8; P < 0.05). Ca2+ wave characteristics at 1 AM K201 suggest a decreased sensitivity of RyR2 to Ca2+; however, 100 AM tetracaine (which decreases RyR2 Ca2+ sensitivity and abolishes Ca2+ waves) led to an enhanced SR Ca2+ content. Assessment of SR Ca2+ ATPase (SERCA) activity indicated a concurrent reduction of both V max and K d. These results suggest that the ability of K201 to reduce the incidence of potentially arrhythmogenic diastolic Ca2+ release is mediated via inhibitory actions on RyR2 and SERCA. doi:10.1016/j.yjmcc.2006.03.034

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020. Phospholamban and troponin I phosphorylation in remodeled left atria from patients with sinus rhythm and chronic atrial fibrillation Ali El-Armouche, Michael Knaut, Thomas Eschenhagen, Ursula Ravens, Dobromir Dobrev Background: Abnormal Ca2+ homeostasis may play an important role in atrial remodeling in atrial fibrillation (AF). Recently, we showed that right atrial appendages of patients with chronic AF have higher protein kinase A (PKA) phosphorylation of phospholamban (PLB) than patients with sinus rhythm (SR). Despite AF originating most frequently from left atria, the molecular mechanisms of impaired left atrial Ca2+ homeostasis are not known. Methods: Here we studied by Western blot with phosphospecific antibodies phosphorylation levels of regulatory PKA substrates in left atrial samples from patients with valvular heart disease (VHD) who were in sinus rhythm (SR-VHD) or in chronic AF (AF-VHD). In order to discriminate between VHD- and AF-related remodeling, left atrial samples from organ donors without structural heart disease were studied as ‘‘normal’’ controls. The troponin inhibitor (TnI) was chosen as a regulator of myofilament Ca2+ sensitivity, phospholamban (PLB) as a major regulator of Ca2+-loading of the sarcoplasmic reticulum. Results: The PKA phosphorylation-level of TnI and PLB (expressed as phospho/total signals) was higher in AF-VHD than in SR-VHD (TnI: 1.1 T 0.1, n = 8, vs. 0.7 T 0.1, n = 7; P < 0.05, PLB: 1.5 T 0.3, n = 8, vs. 0.7 T 0.1, n = 7; P < 0.05). Interestingly, phosphorylation-level of TnI and PLB in donor atria was higher than in SR-VHD but actually not different from that in AF-VHD (TnI: 1.1 T 0.1, n = 5; P < 0.05 vs. SR-VHD; PLB: 1.8 T 0.1, n = 5, P < 0.05 vs. SR-VHD). No significant differences were detected in the CaMKII-dependent Thr17 phosphorylation of PLB between the 3 groups. Conclusions: In summary, PLB- and TnI phosphorylation levels in remodeled left atria with AF were not increased above normal when compared to unaffected controls, but decreased in remodeled left atria with SR. This suggests a potentially protective mechanism of desensitization of the PKA signaling cascade in atrial remodeling. doi:10.1016/j.yjmcc.2006.03.035

021. The calcium receptor and calcium homeostasis in cardiovascular disease Jacob Tfelt-Hansen, Sanela Smajilovic. Laboratory of Molecular Cardiology, Department of Cardiology, Copenhagen University Hospital, Rigshospitalet, Denmark. Copenhagen Heart Arrhythmia Research Center (CHARC), Copenhagen, Denmark Calcium is the most important signal molecule in the cardiovascular system. Calcium is known as a second messenger mediating various physiological actions through calcium chan-