Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT ( P KC- I nteracting C ousin O f T hioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca ²⁺ handing analysis revealed that increases in myofilament Ca ²⁺ responsiveness, together with increased rate of sarcoplasmic reticulum Ca ²⁺ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.

In atrial myocytes immunocytochemistry has shown two groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs) and those at the cell interior not associated with DHPRs. The extent to which the two sets of RyRs are controlled by Ca²⁺ current (I_Ca) or Ca²⁺ diffusion remains to be determined. Here, using rapid (240 Hz) two‐dimensional confocal Ca²⁺ imaging in rat atrial myocytes, we examine directly the role of I_Ca on the two‐dimensional patterns of local and focal Ca²⁺ releases. I_Ca evoked peripheral Ca²⁺ release within 1–4 ms, causing a rapid monophasic local rise of Ca²⁺, which then propagated into the cell interior along sarcomeric lines (∼2 μm) with a velocity of ∼230 μm s⁻¹, even though we found no evidence for organized t‐tubules using di‐8‐ANEPPS staining. I_Ca‐triggered Ca²⁺ release in the cell centre, on the other hand, had both a rapid (≤12 ms) and slower delayed components (12–50 ms). The voltage dependence of peripheral Ca²⁺ release and the two components of central release was bell shaped, and the magnitude of each release component was linearly related to I_Ca. Premature termination (2–10 ms) of I_Ca was equally effective in abbreviating both the peripheral and slow central Ca²⁺ release. High concentration of Ca²⁺ buffers (2–5 mm EGTA plus 1 mm fluo‐3) completely abolished the I_Ca‐gated propagation wave and the slow delayed component of Ca²⁺ release, but had little or no effect on the rapid component of central release. The efficacy of I_Ca to trigger Ca²⁺ release in periphery of the myocyte was ∼5 times higher than in the centre, consistent with the smaller measured central Ca²⁺ release. The quantification of central Ca²⁺ release as a function of peripheral release suggests a cooperative gating mechanism(s) for central release. These findings indicate that both I_Ca and diffusion of Ca²⁺ from the peripheral sites contribute to the gating of Ca²⁺ release from central SR. How in fact the I_Ca‐dependent fast component of central release is activated remains to be determined.

Atrial myocytes, lacking t‐tubules, have two functionally separate groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs), and those at the cell interior not associated with DHPRs. We have previously shown that the Ca²⁺ current (I_Ca)‐gated central Ca²⁺ release has a fast component that is followed by a slower and delayed rising phase. The mechanisms that regulate the central Ca²⁺ releases remain poorly understood. The fast central release component is highly resistant to dialysed Ca²⁺ buffers, while the slower, delayed component is completely suppressed by such exogenous buffers. Here we used dialysis of Ca²⁺ buffers (EGTA) into voltage‐clamped rat atrial myocytes to isolate the fast component of central Ca²⁺ release and examine its properties using rapid (240 Hz) two‐dimensional confocal Ca²⁺ imaging. We found two populations of rat atrial myocytes with respect to the ratio of central to peripheral Ca²⁺ release (R_c/p). In one population (‘group 1’, ∼60% of cells), R_c/p converged on 0.2, while in another population (‘group 2’, ∼40%), R_c/p had a Gaussian distribution with a mean value of 0.625. The fast central release component of group 2 cells appeared to result from in‐focus Ca²⁺ sparks on activation of I_Ca. In group 1 cells intracellular membranes associated with t‐tubular structures were never seen using short exposures to membrane dyes. In most of the group 2 cells, a faint intracellular membrane staining was observed. Quantification of caffeine‐releasable Ca²⁺ pools consistently showed larger central Ca²⁺ stores in group 2 and larger peripheral stores in group 1 cells. The R_c/p was larger at more positive and negative voltages in group 1 cells. In contrast, in group 2 cells, the R_c/p was constant at all voltages. In group 1 cells the gain of peripheral Ca²⁺ release sites (Δ[Ca²⁺]/I_Ca) was larger at −30 than at +20 mV, but significantly dampened at the central sites. On the other hand, the gains of peripheral and central Ca²⁺ releases in group 2 cells showed no voltage dependence. Surprisingly, the voltage dependence of the fast central release component was bell‐shaped and similar to that of I_Ca in both cell groups. Removal of extracellular Ca²⁺ or application of Ni²⁺ (5 mm) suppressed equally I_Ca and Ca²⁺ release from the central release sites at +60 mV. Depolarization to +100 mV, where I_Ca is absent and the Na⁺–Ca²⁺ exchanger (NCX) acts in reverse mode, did not trigger the fast central Ca²⁺ releases in either group, but brief reduction of [Na⁺]_o to levels equivalent to [Na⁺]_i facilitated fast peripheral and central Ca²⁺ releases in group 2 myocytes, but not in group 1 myocytes. In group 2 cells, long‐lasting (> 1 min) exposures to caffeine (10 mm) or ryanodine (20 μm) significantly suppressed I_Ca‐triggered central and peripheral Ca²⁺ releases. Our data suggest significant diversity of local Ca²⁺ signalling in rat atrial myocytes. In one group, I_Ca‐triggered peripheral Ca²⁺ release propagates into the interior triggering central Ca²⁺ release with significant delay. In a second group of myocytes I_Ca triggers a significant number of central sites as rapidly and effectively as the peripheral sites, thereby producing more synchronized Ca²⁺ releases throughout the myocytes. The possible presence of vestigial t‐tubules and larger Ca²⁺ content of central sarcoplasmic reticulum (SR) in group 2 cells may be responsible for the rapid and strong activation of central release of Ca²⁺ in this subset of atrial myocytes.

Atrial myocytes have two functionally separate Ca ²⁺ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca ²⁺ channels of surface membrane and those in central SR not associated with Ca ²⁺ channels. Recently, we have reported on the gating of these two different Ca ²⁺ release sites by Ca ²⁺ current. In the present study, we report on the spatiotemporal properties of focal Ca ²⁺ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca ²⁺ imaging. Peripheral and central sparks were similar in size and release time (≈300 000 Ca ²⁺ ions for ≅12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd ²⁺ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had ≈5-fold higher frequency, ≈2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca ²⁺ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca ²⁺ signaling diversity. The full text of this article is available at http://www.circresaha.org.