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Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP 3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP 3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% ( p = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 μM PE in the presence and absence of 1 μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP 3 R calcium release.

Cardiac hypertrophy increases the risk of morbidity and mortality of cardiovascular disease and thus inhibiting such hypertrophy is beneficial. In the present study, we explored the effect of a bioactive peptide (PAP) on angiotensin II (Ang II)-induced hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models. PAP enhances p21 activated kinase 1 (Pak1) activity by increasing the level of phosphorylated Pak1 in cultured neonatal rat ventricular myocytes (NRVMs). Such PAP-induced Pak1 activation is associated with a significant reduction of Ang II-induced hypertrophy in NRVMs and C57BL/6 mice, in vitro and in vivo, respectively. Furthermore, PAP antagonizes ventricular arrhythmias associated with Ang II-induced hypertrophy in mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pak1 activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt ) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt + cell derived cells) within the heart. Here, we show that Pnmt + cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt + cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt + cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies.

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP 3 ) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP 3 receptors (IR 3 Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP 3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP 3 -evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP 3 Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.

The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac “funny” (f) channels, I(f), and/or suppression of sarcoplasmic reticulum (SR) function in the absence and presence of β‐adrenoceptor stimulation. ZD7288 [to block I(f)] caused a substantial reduction (222 ± 13 bpm) in beating rate from 431 ± 14 to 209 ± 14 bpm, ryanodine alone (to block SR Ca2+ release) reduced beating rate by 105 ± 11 bpm, with subsequent addition of ZD7288 further reducing rate by 57 ± 9 bpm. Cyclopiazonic acid (CPA) alone (to inhibit Ca2+ reuptake by the SR) reduced beating rate by 148 ± 13 bpm with subsequent addition of ZD7288 further reducing rate by 79 ± 12 bpm. In additional experiments measuring Ca2+ transients in the SA node region using Rhod‐2, effects of ivabradine and ZD7288 on rate were again substantially reduced after CPA. Effects of CPA alone on rate developed much more slowly than effects on Ca2+ transient amplitude. ZD7288, ivabradine, and CPA reduced the slope and maximum response of the log(concentration)–response curves for effects of isoprenaline on beating rate. Very little response to isoprenaline remained after treatment with CPA followed by ZD7288. Similar changes in isoprenaline log(concentration)–response curves were seen in guinea pig preparations. These observations are consistent with a role for Ca2+ released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR‐derived Ca2+ to effects of β‐adrenoceptor stimulation on beating rate that are independent of I(f). The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac “funny” (f) channels, I(f), and/or suppression of sarcoplasmic reticulum (SR) function in the absence and presence of β‐adrenoceptor stimulation. The observations are consistent with a role for Ca2+ released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR‐derived Ca2+ to effects of β‐adrenoceptor stimulation on beating rate that are independent of I(f).

Multi-parametric electrophysiological measurements using optical methods have become a highly valued standard in cardiac research. Most published optical mapping systems are expensive and complex. Although some applications demand high-cost components and complex designs, many can be tackled with simpler solutions. Here, we describe (1) a camera-based voltage and calcium imaging system using a single ‘economy’ electron-multiplying charge-coupled device camera and demonstrate the possibility of using a consumer camera for imaging calcium transients of the heart, and (2) a photodiode-based voltage and calcium high temporal resolution measurement system using single-element photodiodes and an optical fibre. High-throughput drug testing represents an application where system scalability is particularly attractive. Therefore, we tested our systems on tissue exposed to a well-characterized and clinically relevant calcium channel blocker, nifedipine, which has been used to treat angina and hypertension. As experimental models, we used the Langendorff-perfused whole-heart and thin ventricular tissue slices, a preparation gaining renewed interest by the cardiac research community. Using our simplified systems, we were able to monitor simultaneously the marked changes in the voltage and calcium transients that are responsible for the negative inotropic effect of the compound.

The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac “funny” (f) channels, I(f), and/or suppression of sarcoplasmic reticulum (SR) function in the absence and presence of β‐adrenoceptor stimulation. ZD7288 [to block I(f)] caused a substantial reduction (222 ± 13 bpm) in beating rate from 431 ± 14 to 209 ± 14 bpm, ryanodine alone (to block SR Ca2+ release) reduced beating rate by 105 ± 11 bpm, with subsequent addition of ZD7288 further reducing rate by 57 ± 9 bpm. Cyclopiazonic acid (CPA) alone (to inhibit Ca2+ reuptake by the SR) reduced beating rate by 148 ± 13 bpm with subsequent addition of ZD7288 further reducing rate by 79 ± 12 bpm. In additional experiments measuring Ca2+ transients in the SA node region using Rhod‐2, effects of ivabradine and ZD7288 on rate were again substantially reduced after CPA. Effects of CPA alone on rate developed much more slowly than effects on Ca2+ transient amplitude. ZD7288, ivabradine, and CPA reduced the slope and maximum response of the log(concentration)–response curves for effects of isoprenaline on beating rate. Very little response to isoprenaline remained after treatment with CPA followed by ZD7288. Similar changes in isoprenaline log(concentration)–response curves were seen in guinea pig preparations. These observations are consistent with a role for Ca2+ released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR‐derived Ca2+ to effects of β‐adrenoceptor stimulation on beating rate that are independent of I(f).

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP 3 ) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP 3 receptors (IR 3 Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP 3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP 3 -evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP 3 Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.