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p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1(-/-) mice. Pak1(-/-) Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased T-tubular density in Pak1(-/-) VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1(-/-) mice where the T-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the T-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the T-tubular system whose remodeling is an integral feature of hypertrophic remodeling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Since techniques for cardiomyocyte isolation were first developed 35 years ago, experiments on single myocytes have yielded great insight into their cellular and sub-cellular physiology. These studies have employed a broad range of techniques including electrophysiology, calcium imaging, cell mechanics, immunohistochemistry and protein biochemistry. More recently, techniques for cardiomyocyte culture have gained additional importance with the advent of gene transfer technology. While such studies require a high quality cardiomyocyte population, successful cell isolation and maintenance during culture remain challenging. In this review, we describe methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart. This discussion outlines general principles for the beginner, but also provides detailed specific protocols and advice for common caveats. We additionally review methods for short-term myocyte culture, with particular attention given to the importance of substrate and media selection, and describe time-dependent alterations in myocyte physiology that should be anticipated. Gene transfer techniques for neonatal and adult cardiomyocytes are also reviewed, including methods for transfection (liposome, electroporation) and viral-based gene delivery., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although bradykinin (BK) is known to exert effects on the myocardium, its intracellular signaling pathways remain poorly understood. Experiments in other cell types indicated that p21-activated kinase-1 (Pak1), a Ser/Thr kinase downstream of small monomeric G proteins, is activated by BK. We previously reported that the expression of active Pak1 in adult cardiac myocytes induced activation of protein phosphatase 2A and dephosphorylation of myofilament proteins (Ke et al. Circ Res 94: 194-200, 2004). In experiments reported here, we tested the hypothesis that BK signals altered protein phosphorylation in adult rat cardiac myocytes through the activation and translocation of Pak1. Treatment of myocytes with BK resulted in the activation of Pak1 as demonstrated by increased autophosphorylation at Thr423 and a diminished striated localization, which is present in the basal state. BK induced dephosphorylation of both cardiac troponin I and phospholamban. Treatment of isolated myocytes with BK also blunted the effect of isoproterenol to enhance peak Ca(2+) and relaxation of Ca(2+) transients. Protein phosphatase 2A was demonstrated to associate with both Pak 1 and phospholamban. Our studies indicate a novel signaling mechanism for BK in adult rat cardiac myocytes and support our hypothesis that Pak 1 is a significant regulator of phosphatase activity in the heart.

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.

Calcium sparks result from the concerted opening of a small number of Ca2+ release channels (ryanodine receptors, RyRs) organized in clusters in the membrane of the sarcoplasmic reticulum (SR). Calcium sparks represent the elementary events of SR Ca2+ release in cardiac myocytes, and their spatial and temporal summation results in whole-cell [Ca2+]i transients observed during excitation-contraction coupling (ECC). Atrial myocytes generally lack transverse tubules; however, during ECC Ca2+ release is initiated from junctional SR (j-SR) in the cell periphery from where activation propagates inwardly through Ca(2+)-induced Ca2+ release (CICR) from non-junctional SR (nj-SR). Despite the structural differences in the microdomains of RyRs of j-SR and nj-SR, spontaneous Ca2+ sparks are observed from both types of SR, albeit at different frequencies. In cells that showed spontaneous Ca2+ sparks from j-SR and nj-SR, subsarcolemmal (SS) Ca2+ sparks from the j-SR were 3-4 times more frequent than central (CTR) Ca2+ sparks occurring from nj-SR. Subsarcolemmal Ca2+ sparks had a slightly higher amplitude, but were essentially identical in their spatial spread and duration when compared to CTR Ca2+ sparks. Sensitization of RyRs with a low concentration (0.1 mM) of caffeine led to a 107% increase in the frequency of CTR Ca2+ sparks, whereas the SS Ca2+ spark frequency increased by only 58%, suggesting that the nj-SR is capable of much higher Ca2+ spark activity than observed normally in unstimulated cells. The L-type Ca2+ channel blocker verapamil reduced SS Ca2+ spark frequency to 38% of control values, whereas Ca2+ spark activity from nj-SR was reduced by only 19%, suggesting that SS Ca2+ sparks are under the control of Ca2+ influx from the extracellular space. Removal of extracellular Ca2+ eliminated SS Ca2+ sparks completely, whereas Ca2+ sparks from the nj-SR continued, albeit at a lower frequency. In membrane-permeabilized (saponin-treated) atrial myocytes, where [Ca2+] can be experimentally controlled throughout the entire myocyte, j-SR and nj-SR Ca2+ spark frequencies were identical, and Ca2+ sparks could be observed spaced at sarcomeric distances throughout the entire cell, suggesting that all release sites of the nj-SR can become active. Measurement of SR Ca2+ load (10 mM caffeine) revealed no difference between j-SR and nj-SR. The data suggest that in atrial myocytes, which lack a t-tubular system, the nj-SR is fully equipped with a three-dimensional array of functional SR Ca2+ release sites; however, in intact cells under resting conditions, peripheral RyR clusters have a higher probability of activation owing to their association with surface membrane Ca2+ channels, leading to higher spontaneous Ca2+ spark activity. In conclusion, Ca2+ sparks originating from both j-SR and nj-SR are rather stereotypical and show little differences in their spatiotemporal properties. In intact cells, however, the higher frequency of spontaneous SS Ca2+ sparks arises from the structural arrangement of sarcolemma and j-SR membrane and thus from the difference in the trigger mechanism.

Palytoxin is a coral toxin that seriously impairs heart function, but its effects on excitation-contraction (E-C) coupling have remained elusive. Therefore, we studied the effects of palytoxin on mechanisms involved in atrial E-C coupling. In field-stimulated cat atrial myocytes, palytoxin caused elevation of diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)), a decrease in [Ca(2+)](i) transient amplitude, Ca(2+) alternans followed by [Ca(2+)](i) waves, and failures of Ca(2+) release. The decrease in [Ca(2+)](i) transient amplitude occurred despite high sarcoplasmic reticulum (SR) Ca(2+) load. In voltage-clamped myocytes, palytoxin induced a current with a linear current-voltage relationship (reversal potential approximately 5 mV) that was blocked by ouabain. Whole cell Ca(2+) current and ryanodine receptor Ca(2+) release channel function remained unaffected by the toxin. However, palytoxin significantly reduced Ca(2+) pumping of isolated SR vesicles. In current-clamped myocytes stimulated at 1 Hz, palytoxin induced a depolarization of the resting membrane potential that was accompanied by delayed afterdepolarizations. No major changes of action potential configuration were observed. The results demonstrate that palytoxin interferes with the function of the sarcolemmal Na(+)-K(+) pump and the SR Ca(2+) pump. The suggested mode of palytoxin toxicity in the atrium involves the conversion of Na(+)-K(+) pumps into nonselective cation channels as a primary event followed by depolarization, Na(+) accumulation, and Ca(2+) overload, which, in turn, causes arrhythmogenic [Ca(2+)](i) waves and delayed afterdepolarizations.

The inotropic and toxic effects of cardiac glycosides are thought to be related to their ability to inhibit the Na,K-ATPase. We examined the effects of ouabain and its analogs on sarcoplasmic reticulum (SR) Ca(2+) release in intact cat ventricular myocytes under Na(+)-free conditions and in myocytes in which the sarcolemma was permeabilized using saponin so that cytoplasmic ionic composition was fixed by the bath solutions. We also compared ouabain actions in cat myocytes to those in rat myocytes because the latter is considered to be a glycoside-insensitive species. In intact cat myocytes (Na(+)-free conditions), spontaneous Ca(2+) sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 microM). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 microM). The frequency of spontaneous Ca(2+) waves was also reduced by ouabain. In contrast, ouabain (100 microM) had negligible effects on sparks and waves in rat myocytes in Na(+)-free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (>or=50 nM) decreased spark frequency and increased background SR Ca(2+) leak only when the SR was well loaded (free [Ca(2+)] = 275 nM) and not when SR load was low (free [Ca(2+)] = 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 microM. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca(2+) release, possibly through activation of SR Ca(2+) release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca(2+) release is dependent on the extent of SR Ca(2+) load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

We have characterized the dependence on membrane potential (V(m)) and calcium current (I(Ca)) of calcium-induced calcium release (CICR) from the junctional-SR (j-SR, in the subsarcolemmal (SS) space) and non-junctional-SR (nj-SR, in the central (CT) region of the cell) of cat atrial myocytes using whole-cell voltage-clamp together with spatially resolved laser-scanning confocal microscopy. Subsarcolemmal and central [Ca(2+)](i) transient amplitudes and I(Ca) had a bell-shaped dependence on V(m), but [Ca(2+)](i) reached a maximum at more negative V(m) (-10 to 0 mV) than I(Ca) (+10 mV). Termination of I(Ca) after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca(2+)](i) transient, leaving the development of the CT [Ca(2+)](i) transient unaffected. Block of SR function with 20 microM ryanodine and 2 microM thapsigargin, revealed that > 90 % of the control [Ca(2+)](i) transient amplitude was attributable to active SR Ca(2+) release through ryanodine receptors (RyRs). The gain of SR Ca(2+) release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na(+)-Ca(2+) exchange resulted in prolonged and higher amplitude [Ca(2+)](i) transients, elevated resting [Ca(2+)](i), accelerated propagation of CICR, decreased extrusion of Ca(2+) and an increase in j-SR Ca(2+) load. Increasing the cytosolic Ca(2+) buffer capacity by internal perfusion with 1 mM EGTA limited SR Ca(2+) release to the SS region, indicating that Ca(2+) release from nj-SR is initiated by diffusion of Ca(2+) from the cell periphery and propagating CICR. Junctional-SR Ca(2+) release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation-contraction coupling in cat atrial myocytes, only Ca(2+) release from the j-SR is directly activated by Ca(2+) entering via I(Ca). Elevation of SS [Ca(2+)](i) is required to provide the cytosolic Ca(2+) gradient needed to initiate regenerative and propagating CICR from nj-SR.

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