Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease.

Inositol 1,4,5-trisphosphate (IP 3 ) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP 3 from phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The major action of IP 3 , which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca 2+ release from endogenous stores through IP 3 receptors (IP 3 Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca 2+ release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP 3 Rs (∼100:1), and furthermore they experience substantial fluxes of Ca 2+ with each heartbeat. Therefore, the role of IP 3 and IP 3 -mediated Ca 2+ signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP 3 Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP 3 Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca 2+ signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP 3 Rs and membrane receptors that couple to PLC/IP 3 signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP 3 signaling in the pathology of these diseases. Thus, IP 3 exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure. KeywordsInositol 1,4,5-trisphosphate; Cardiac myocyte; Calcium; Inotropy; Arrhythmias; Nucleus; Hypertrophy © 2008 Elsevier Inc. All rights reserved. * Corresponding author. E-mail address: jens.kockskaemper@meduni-graz.at (J. Kockskämper).. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript The discovery of IP 3A quarter of a century ago it was shown that D-myo inositol 1,4,5-trisphosphate (IP 3 ) releases Ca 2+ from a non-mitochondrial internal Ca 2+ store [1]. Since this hallmark discovery, IP 3 has emerged as a ubiquitous intracellular messenger, releasing Ca 2+ from stores through activation of IP 3 receptors (IP 3 Rs) in almost all eukaryotic cells. The major IP 3 -sensitive intracellular Ca 2+ store is the endoplasmic reticulum. However, IP 3 has also been shown to release Ca 2+ stored in other compartments, such as the Golgi and the nuclear envelope [2]. In addition, IP 3 Rs are present on the plasma membrane of some cell types, where they can gate Ca 2+ influx [3]. A crucial role for IP 3 -dependent Ca 2+ release has been demonstrated in many mammalian cell types, ranging from tiny platelets, where it initiates blood clottin...

Subcellular Ca2+ signalling during normal excitation‐contraction (E‐C) coupling and during Ca2+ alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca2+ currents (ICa). Ca2+ alternans, a beat‐to‐beat alternation in the amplitude of the [Ca2+]i transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j‐SR) and non‐junctional (nj‐SR) types, both of which have ryanodine‐receptor calcium release channels. During E‐C coupling, Ca2+ entering through voltage‐gated membrane Ca2+ channels (ICa) triggers Ca2+ release at discrete peripheral j‐SR release sites. The discrete Ca2+ spark‐like increases of [Ca2+]i then fuse into a peripheral ‘ring’ of elevated [Ca2+]i, followed by propagation (via calcium‐induced Ca2+ release, CICR) to the cell centre, resulting in contraction. Interrupting ICa instantaneously terminates j‐SR Ca2+ release, whereas nj‐SR Ca2+ release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca2+ alternans. The spatiotemporal [Ca2+]i pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca2+]i and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca2+ alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca2+ waves develop at their border. In conclusion, the results demonstrate that (1) during normal E‐C coupling the atrial [Ca2+]i transient is the result of the spatiotemporal summation of Ca2+ release from individual release sites of the peripheral j‐SR and the central nj‐SR, activated in a centripetal fashion by CICR via ICa and Ca2+ release from j‐SR, respectively, (2) Ca2+ alternans is caused by subcellular alterations of SR Ca2+ release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca2+ waves resulting from heterogeneities in subcellular Ca2+ alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.