- Email: [email protected]
The calcium receptor and calcium homeostasis in cardiovascular disease
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
927
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-
928
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
nels and pumps. However, our results and those from other investigators have shown that calcium seems to exert some of its actions through the calcium receptor (CaR). The calcium receptor belongs to the super family C of G-protein coupled receptors. The receptor is mainly present in organs involved in calcium homoeostasis such as the parathyroid and kidney. Recent studies showed that the CaR is also present in the endothelial cells of mesenteric and coronary arteries as well as aorta. Stimulating the CaR with a positive allosteric CaR modulator induced a hyperpolarisation of vascular smooth muscle cells in the mesenteric and coronary arteries suggesting a role of the CaR in the control of vascular tone. In isolated rat neonatale cardiomyocytes (NCM), we found the CaR to be present on the messenger RNA and protein level. We found that stimulating the CaR in rat NCM induced production of inositol phosphate and activation of mitogenic-activated protein kinase, two well described pathway downstream of the receptor. Calcium and AMG073, a positive allosteric modulator of the CaR, decreased the DNA synthesis. To summarise, the current knowledge of the CaR in the cardiovascular system: The CaR is functionally expressed in the cardiomyocytes, endothelial cells of the mesenteric and coronary arteries, two major players in the cardiovascular system. Our results suggest that CaR downregulates DNA synthesis of the myocardium and data from others suggest that the CaR regulates the contraction of the VSMC in the mesenteric and coronary arteries. Future studies will show the importance of the receptor in the normal physiology as well as the possible roles in the pathophysiology of disease in the cardiovascular system such as hypertension and arteriosclerosis. doi:10.1016/j.yjmcc.2006.03.036
022. Endogenous inhibitors of nitric oxide synthesis: How important are they? Manasi Nandi, James Leiper, Patrick Vallance. Centre for Clinical Pharmacology, Division of Medicine, University College London, UK
023. The progression of contractile dysfunction correlates with the extent of myofibrillar protein oxidation in failing rabbit hearts Marcella Canton a, Stephanie Aker b, Roberta Menabo` a, Gerd Heusch b, Fabio Di Lisa a, Rainer Schulz b. a CNR, University of Padova, Padova, Italy. b University of Essen, Essen, Germany Although ROS production is increased in heart failure (HF), scarce information is available whether ROS-induced oxidative processes might affect the structure and function of myofibrillar proteins (MP) in HF. Therefore, we investigated the occurrence of MP oxidation in HF assessing the correlation with the progression of contractile dysfunction. We have recently demonstrated that tropomyosin (TM) undergoes reversible oxidation as shown by the formation of disulfide cross-bridges that are only present in immunoblots after non-reducing SDS-PAGE (Eur Heart J 2006 Jan 31). This modification is reflected by immunoreactive changes that are blunted by adding dithiothreitol, a thiol reducing agent, to cryosections. In the present study, MP oxidation was assessed as an increase in TM immunofluorescence (IF). HF was induced by left ventricular pacing (400 bpm) for 1 week (n = 6), 2 weeks (n = 6) or 3 weeks (n = 9). Eight sham-operated rabbits served as controls. IF values of the paced groups were normalized to the mean average of samples from sham animals. With increased duration of pacing, shortening fraction (SF,%) progressively deteriorated from 30.9 T 0.9 in shams to 19.1 T 0.7, 16.9 T 1.3, and 10.6 T 1.3 after 1, 2 and 3 weeks, respectively. IF values were not different in samples from sham and 1 week paced rabbits, whereas samples from 2 and 3 week paced rabbits displayed a significant increase (1.37 and 1.41, respectively) that was homogenously distributed. The IF increase correlated negatively with %SF (r = 0.62). In conclusion, the present results provide the first direct evidence of a correlation between MP oxidation and contractile dysfunction in failing hearts. doi:10.1016/j.yjmcc.2006.03.038
Plasma levels of an endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), are elevated in a number of disease states including chronic renal failure, pulmonary hypertension, and chronic heart failure. In patients with renal failure, plasma ADMA levels are an independent correlate of left ventricular ejection fraction. However, a causal role for ADMA in the development of cardiovascular disease remains to be demonstrated. In order to test the hypothesis that ADMA has a causal role in disease, we have undertaken molecular and pharmacological approaches to create ex vivo and in vivo systems in which ADMA concentrations are elevated. The role of ADMA in the regulation of vascular function in these model systems will be discussed. doi:10.1016/j.yjmcc.2006.03.037
024. Direct activation of Type I PKA by oxidants independently of cAMP is mediated by RI subunit interprotein disulphide bond formation Jonathan P. Brennan, Sonya C. Bardswell, Joseph R. Burgoyne, William Fuller, Ewald Schro¨der, Jonathan C. Kentish, Philip Eaton. King’s College London, St Thomas’ Hospital, London, UK We have been studying the post-translational modification of protein cysteinyl thiols as a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. In these studies, we demonstrate that Type I protein kinase A is redox active, forming an interprotein disulphide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. Isolated rat hearts
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
927
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-
928
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
nels and pumps. However, our results and those from other investigators have shown that calcium seems to exert some of its actions through the calcium receptor (CaR). The calcium receptor belongs to the super family C of G-protein coupled receptors. The receptor is mainly present in organs involved in calcium homoeostasis such as the parathyroid and kidney. Recent studies showed that the CaR is also present in the endothelial cells of mesenteric and coronary arteries as well as aorta. Stimulating the CaR with a positive allosteric CaR modulator induced a hyperpolarisation of vascular smooth muscle cells in the mesenteric and coronary arteries suggesting a role of the CaR in the control of vascular tone. In isolated rat neonatale cardiomyocytes (NCM), we found the CaR to be present on the messenger RNA and protein level. We found that stimulating the CaR in rat NCM induced production of inositol phosphate and activation of mitogenic-activated protein kinase, two well described pathway downstream of the receptor. Calcium and AMG073, a positive allosteric modulator of the CaR, decreased the DNA synthesis. To summarise, the current knowledge of the CaR in the cardiovascular system: The CaR is functionally expressed in the cardiomyocytes, endothelial cells of the mesenteric and coronary arteries, two major players in the cardiovascular system. Our results suggest that CaR downregulates DNA synthesis of the myocardium and data from others suggest that the CaR regulates the contraction of the VSMC in the mesenteric and coronary arteries. Future studies will show the importance of the receptor in the normal physiology as well as the possible roles in the pathophysiology of disease in the cardiovascular system such as hypertension and arteriosclerosis. doi:10.1016/j.yjmcc.2006.03.036
022. Endogenous inhibitors of nitric oxide synthesis: How important are they? Manasi Nandi, James Leiper, Patrick Vallance. Centre for Clinical Pharmacology, Division of Medicine, University College London, UK
023. The progression of contractile dysfunction correlates with the extent of myofibrillar protein oxidation in failing rabbit hearts Marcella Canton a, Stephanie Aker b, Roberta Menabo` a, Gerd Heusch b, Fabio Di Lisa a, Rainer Schulz b. a CNR, University of Padova, Padova, Italy. b University of Essen, Essen, Germany Although ROS production is increased in heart failure (HF), scarce information is available whether ROS-induced oxidative processes might affect the structure and function of myofibrillar proteins (MP) in HF. Therefore, we investigated the occurrence of MP oxidation in HF assessing the correlation with the progression of contractile dysfunction. We have recently demonstrated that tropomyosin (TM) undergoes reversible oxidation as shown by the formation of disulfide cross-bridges that are only present in immunoblots after non-reducing SDS-PAGE (Eur Heart J 2006 Jan 31). This modification is reflected by immunoreactive changes that are blunted by adding dithiothreitol, a thiol reducing agent, to cryosections. In the present study, MP oxidation was assessed as an increase in TM immunofluorescence (IF). HF was induced by left ventricular pacing (400 bpm) for 1 week (n = 6), 2 weeks (n = 6) or 3 weeks (n = 9). Eight sham-operated rabbits served as controls. IF values of the paced groups were normalized to the mean average of samples from sham animals. With increased duration of pacing, shortening fraction (SF,%) progressively deteriorated from 30.9 T 0.9 in shams to 19.1 T 0.7, 16.9 T 1.3, and 10.6 T 1.3 after 1, 2 and 3 weeks, respectively. IF values were not different in samples from sham and 1 week paced rabbits, whereas samples from 2 and 3 week paced rabbits displayed a significant increase (1.37 and 1.41, respectively) that was homogenously distributed. The IF increase correlated negatively with %SF (r = 0.62). In conclusion, the present results provide the first direct evidence of a correlation between MP oxidation and contractile dysfunction in failing hearts. doi:10.1016/j.yjmcc.2006.03.038
Plasma levels of an endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), are elevated in a number of disease states including chronic renal failure, pulmonary hypertension, and chronic heart failure. In patients with renal failure, plasma ADMA levels are an independent correlate of left ventricular ejection fraction. However, a causal role for ADMA in the development of cardiovascular disease remains to be demonstrated. In order to test the hypothesis that ADMA has a causal role in disease, we have undertaken molecular and pharmacological approaches to create ex vivo and in vivo systems in which ADMA concentrations are elevated. The role of ADMA in the regulation of vascular function in these model systems will be discussed. doi:10.1016/j.yjmcc.2006.03.037
024. Direct activation of Type I PKA by oxidants independently of cAMP is mediated by RI subunit interprotein disulphide bond formation Jonathan P. Brennan, Sonya C. Bardswell, Joseph R. Burgoyne, William Fuller, Ewald Schro¨der, Jonathan C. Kentish, Philip Eaton. King’s College London, St Thomas’ Hospital, London, UK We have been studying the post-translational modification of protein cysteinyl thiols as a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. In these studies, we demonstrate that Type I protein kinase A is redox active, forming an interprotein disulphide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. Isolated rat hearts