Abstract-Ca2ϩ /calmodulin-dependent protein kinase II (CaMKII) ␦ is the predominant cardiac isoform, and the ␦ C splice variant is cytoplasmic. We overexpressed CaMKII␦ C in mouse heart and observed dilated heart failure and altered myocyte Ca 2ϩ regulation in 3-month-old CaMKII␦ C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca 2ϩ ] i transient amplitude, and diastolic [Ca 2ϩ ] i were all reduced by Ϸ50% in TG versus WT. This is explained by Ͼ50% reduction in SR Ca 2ϩ content in TG versus WT. Peak Ca 2ϩ current (I Ca ) was slightly increased, and action potential duration was prolonged in TG versus WT. CaMKII was initially identified in the nervous system, but is found in most tissues, including heart. 3,4 CaMKII␦ is the predominant isoform in heart. 3,5 CaMKII phosphorylates several Ca 2ϩ transport proteins, including ryanodine receptors (RyRs) 6,7 and phospholamban (PLB). 8,9 CaMKII is involved in L-type Ca 2ϩ current (I Ca ) facilitation 10,11 and frequency-dependent acceleration of relaxation (FDAR; which depends on SR Ca 2ϩ uptake). 12-16 Ramirez et al 17 found that the nuclear CaMKII isoform (␦ B ) caused transcriptional activation and expression of atrial natriuretic peptide (ANF, a hypertrophic signaling marker) in neonatal rat ventricular myocytes. Transgenic overexpression of CaMKII␦ B or expression of CaMKIV (also nuclear) induces cardiac hypertrophy. 18,19 However, in vivo expression of cytoplasmic CaMKII (␦ C ) has not been examined.In human heart failure (HF), CaMK activity is increased 2-to 3-fold, which could be compensatory because it correlated positively with cardiac index and ejection fraction in patients. 20,21 Because phosphatase activity is also enhanced in human HF, 22 the net phosphorylation state of individual targets is unclear. For example, whereas many protein kinase A (PKA) targets are relatively dephosphorylated in HF, RyRs can be hyperphosphorylated due to reduced local phosphatase bound to RyRs. 23 To investigate CaMKII␦ effects on cellular Ca 2ϩ regulation, we overexpressed cytoplasmic CaMKII␦ C in mouse heart. 24 The transgenic line studied here exhibits 3-fold increase in CaMKII activity, profound dilated hypertrophy, and ventricular dysfunction. 24 Materials and Methods Generating CaMKII␦ C Transgenic Mice and Myocyte IsolationTransgenic mice were generated as in Zhang et al. 24 In the present study, Ca 2ϩ handling was assessed using (unless noted) 3-month-old CaMKII␦ C TG mice (nϭ8) exhibiting a 3-fold increase in CaMKII activity (TGM line 24 ), and age-matched WT littermates (nϭ8). Ventricular myocytes were isolated as reported 14 ] i-rest was used. Results were comparable among indo-1, fluo-3, and fluo-4, so results were pooled. Ca 2ϩ sparks were characterized by an algorithm (IDL 5.3) 29 using a threshold of 3.8ϫSD with human verification. We measured Ca 2ϩ spark peak (F/F 0 ), duration (full-duration-half-maximum, FDHM), width, or spa...

Abstract-Cardiac 4 -6 Dysregulation of these processes underlie arrhythmogenesis during ischemia and heart failure and in certain inherited conditions. 7,8 Intracellular Ca 2ϩ alternans is the cellular correlate of cardiac alternans, a beat-to-beat variation in cardiac contractility and repolarization at a constant heart rate. Cardiac alternans occurs during ischemia, acidosis, and heart failure and is a prominent risk factor for the development of ventricular arrhythmias. 9 Materials and MethodsVentricular myocytes were isolated from New Zealand White rabbits and loaded with the membrane-permeable form of Fluo5N under conditions that promote dye accumulation in the SR as previously described. 21 All experiments were performed at room temperature. For [Ca 2ϩ ] SR measurements, fluorescence was recorded on wide-field epifluorescence microscopes at an excitation wavelength of 488 nm and emission of Ͼ500 nm. When [Ca 2ϩ ] SR depletions and cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) transients were measured simultaneously, cells were loaded additionally with Fura2/acetoxymethyl ester (Fura2/AM) and fluorescence was excited sequentially at 340, 380, and 488 nm and emitted light was detected at Ͼ500 nm. Twitches were either evoked by extracellular field stimulation or by stimulating pulses under current-clamp conditions. Cells were continuously superfused with a bath solution consisting of (in mmol/L) CaCl 2 2, NaCl 140, KCl 4, MgCl 2 1, HEPES 5, and glucose 10 (pH 7.4). APs were recorded under current clamp using the perforated patch technique. I Ca was measured under voltage clamp in the ruptured patch configuration.Data were analyzed offline with custom-made software. Diastolic [Ca 2ϩ ] SR refers to [Ca 2ϩ ] SR immediately preceding the beginning of the depletion. The alternans ratio (AR) of depletion amplitudes was calculated as described previously 22 with modification to allow application during reloading of the SR: ARϭ1Ϫ2 S n /(L nϪ1 ϩL nϩ1 ), where S n is the small depletion amplitude and L nϪ1 and L nϩ1 are the large depletion amplitudes preceding and following S n , respectively.Data are expressed as meanϮSD. One-way ANOVA was used to test for statistical significance. An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. (Figure 2A, with sections on an expanded time scale in Figure 2B). Figure 2C shows the relationship between diastolic [Ca 2ϩ ] SR and the immediately following depletion amplitude for the entire trace in Figure 2A. During regular depletions (without alternans) [Ca 2ϩ ] SR depletion amplitudes and diastolic [Ca 2ϩ ] SR were consistent, such that the data points clustered very tightly in a single area of the graph. This illustrates the small beat-to-beat variation and Figure 2B and 2C also indicates the great sensitivity of these [Ca 2ϩ ] SR measurements, where a difference of Ϸ3% in diastolic [Ca 2ϩ ] SR signal is quite easily resolved during alternans ( Figure I in the online data supplement). Results [The upper cu...

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.

Objective: Here, we generated conditional, heart-specific transgenic mice with both gain-and loss-of-function for IP 3 receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation. Methods and Results:Overexpression of the mouse type-2 IP 3 receptor (IP 3 R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca 2؉ transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP 3 R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to G␣q overexpression, and/or in response to exercise stimulation. To inhibit IP 3 signaling in vivo, we generated transgenic mice expressing an IP 3 chelating protein (IP 3 -sponge). IP 3 -sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP 3 R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-A␤-null background. IP 3 is a second messenger generated by hydrolysis of membrane lipid phosphatidyl-inositol 4,5-bisphosphate by phospholipase (PL)C in response to GPCR activation associated with growth factors and neuroendocrine agonists. 6 Once generated, IP 3 causes Ca 2ϩ release from intracellular stores by binding the IP 3 receptor (IP 3 R), an intracellular Ca 2ϩ release channel embedded in the sarcoplasmic reticulum (SR) and nuclear envelope. Cardiac hypertrophy has been associated with increased PLC activity and increased generation of IP 3 . 7,8 Moreover, expression of IP 3 Rs is increased in both human and animal models of heart failure, suggesting that this form of Ca The IP 3 R family consists of 3 genes 11 : IP 3 R1, IP 3 R2, and IP 3 R3. IP 3 R2 is thought to be the most prominent gene expressed in the heart, 12,13 and its deletion in gene-targeted mice abolished positive inotropy and spontaneous Ca 2ϩ release in atrial myocytes caused by endothelin (ET)-1 stimulation. 13 Even though ventricular myocytes express much lower levels of IP 3 Rs than atrial myocytes, these receptors, in some reports, can alter Ca 2ϩ release and predispose to arrhythmia. 14 -16 Although the IP 3 Rs can affect Ca 2ϩ release, it has not been possible to determine their necessity in regulating the cardiac hypertrophic response because all 3 receptor genes are expressed in the heart, complicating a gene-targeting approach, not withstanding lethality issues in IP 3 R1-null mice. Here, we generated transgenic mice with IP 3 R2 overexpression and the inhibitory IP 3 -sponge protein, demonstrating for the first time that the IP 3 R functions as a hypertrophic effector in vivo. Conclusion Methods Generation of Transgenic MicecDNAs encoding mouse IP 3 R2 and recombinant Flag-tagged IP 3 -sponge protein 17,18 were...