Abstract-cAMP is one of the most important second messenger in the heart. The discovery of Epac as a guanine exchange factor (GEF), which is directly activated by cAMP, raises the question of the role of this protein in cardiac cells. Here we show that Epac activation leads to morphological changes and induces expression of cardiac hypertrophic markers. This process is associated with a Ca 2ϩ -dependent activation of the small GTPase, Rac. In addition, we found that Epac activates a prohypertrophic signaling pathway, which involves the Ca 2ϩ sensitive phosphatase, calcineurin, and its primary downstream effector, NFAT. Rac is involved in Epac-induced NFAT dependent cardiomyocyte hypertrophy. Key Words:cAMP Ⅲ guanine nucleotide exchange factor Ⅲ small G protein Ⅲ transcription factor I n the heart, cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) regulates many physiological processes such as contractility and relaxation. Classically, these effects are attributed to activation of hyperpolarization-activated cyclic nucleotidegated channels and protein kinase A (PKA) by cAMP. 1 The recent discovery of Epac as proteins which are directly activated by cAMP has broken the dogma surrounding cAMP and PKA. [2][3][4] Epac proteins are guanine nucleotide exchange factors (GEFs) that bind cAMP with affinities similar to that of the regulatory subunit of PKA. 2,3 They have been shown to function as GEFs for the Ras-like small GTPases Rap1 and Rap2 and are directly activated by cAMP in a PKA independent manner. 4 There are two isoforms of Epac, Epac 1 and Epac 2 both consisting of a regulatory and a catalytic region. 2,3 Epac 2 has an additional cAMP binding domain that is dispensable for cAMP-induced Rap activation. 5 After cAMP binding, Epac catalyzes the exchange of GDP for GTP on the small GTPases Rap, allowing interaction with their target effectors. 6 Recent studies indicate that Epac is involved in cell adhesion, 7,8 neurite extension, 9 and regulates insulin secretion and the amyloid precursor protein processing. 10,11 To date the role of Epac in the heart is unknown.Among the superfamily of small G proteins, the Rho family, which includes Rho, Rac, and Cdc42, has attracted much interest for they have been shown to play key roles in the generation of cytoskeletal structures. 12 Indeed, Rho is important for the formation of stress fibers and focal adhesions in fibroblasts, whereas Rac and Cdc42 are involved in the regulation of more dynamic structures such as membrane ruffles, lamellipodia and filopodia. 12 Several studies have pointed out the role of Rho proteins in the development of cardiomyocyte hypertrophy. 13 For instance, two potent hypertrophic stimuli, endothelin 1 (ET-1) and phenylephrine (PE), induce rapid activation of endogenous Rac in neonatal cardiomyocytes. 14 In addition, adenoviral infection of cardiomyocytes with a constitutive active form of Rac (Rac G12V ) increases protein synthesis and promotes morphological changes associated with myocyte hypertrophy. 15 In vivo evidence for the role of Rho proteins...

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 μm2/s in the longitudinal and 52 ± 16 μm2/s in the transverse directions (n = 8, mean ± SD). Those values are ∼2 (longitudinal) and ∼3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine:glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT knockouts and wild-type littermates as well as in AGAT knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole-cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation was significantly lower in AGAT knockout compared to wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared to wild-type: LTCC conductance, the exchange constant of calcium transfer between subspace and cytosol, and SERCA activity. Since tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of post-translational regulatory mechanisms or structural reorganization. The AGAT knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.

We have developed a novel method to quantitatively analyze mitochondrial positioning in three dimensions. Using this method, we compared the relative positioning of mitochondria in adult rat and rainbow trout (Oncorhynchus mykiss) ventricular myocytes. Energetic data suggest that trout, in contrast to the rat, have two subpopulations of mitochondria in their cardiomyocytes. Therefore, we speculated whether trout cardiomyocytes exhibit two types of mitochondrial patterns. Stacks of confocal images of mitochondria were acquired in live cardiomyocytes. The images were processed and mitochondrial centers were detected automatically. The mitochondrial arrangement was analyzed by calculating the three-dimensional probability density and distribution functions describing the distances between neighboring mitochondrial centers. In the rat (8 cells with a total of 7,546 mitochondrial centers), intermyofibrillar mitochondria are highly ordered and arranged in parallel strands. These strands are separated by approximately 1.8 mum and can be found in any transversal direction relative to each other. Neighboring strands exhibit the same mitochondrial periodicity. In contrast to the rat, trout ventricular myocytes (22 cells; 5,528 mitochondrial centers) exhibit a relatively chaotic mitochondrial pattern. Neighboring mitochondria can be found in any direction relative to each other. Thus, two potential subpopulations of mitochondria in trout are not distinguishable by their pattern. The developed method required minor interaction in the filtering of the mitochondrial centers. It is therefore a practical approach to describe intracellular organization and may also be used for analysis of time-dependent organizational changes. The obtained quantitative description of mitochondrial organization is a requisite for accurate mathematical analysis of mitochondrial systems biology.