Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.

Over-nutrition and insulin resistance are especially prominent risk factors for the development of cardiac diastolic dysfunction in females. We recently reported that consumption of a western diet (WD) containing excess fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) for 16 weeks resulted in cardiac diastolic dysfunction and aortic stiffening in young female mice and that these abnormalities were prevented by mineralocorticoid receptor blockade. Herein, we extend those studies by testing whether WD-induced diastolic dysfunction, and factors contributing to diastolic impairment, such as cardiac fibrosis, hypertrophy, inflammation and impaired insulin signaling, are modulated by excess endothelial cell mineralocorticoid receptor (ECMR) signaling. Four week-old female ECMR knockout and wild type mice were fed mouse chow or WD for 4 months. WD feeding resulted in prolonged relaxation time, impaired diastolic septal wall motion and increased left ventricular (LV) filling pressure indicative of diastolic dysfunction. This occurred in concert with myocardial interstitial fibrosis and cardiomyocyte hypertrophy that was associated with enhanced pro-fibrotic (TGF-β1/Smad) and pro-growth (S6 kinase-1) signaling, as well as myocardial oxidative stress and a pro-inflammatory immune response. WD also induced cardiomyocyte stiffening, assessed ex vivo using atomic force microscopy. Conversely, ECMR deficiency prevented WD-induced diastolic dysfunction, pro-fibrotic and pro-growth signaling, in conjunction with reductions in macrophage pro-inflammatory polarization and improvements in insulin metabolic signaling. Therefore, our findings indicate that increased ECMR signaling associated with consumption of a WD plays a key role in activation of cardiac pro-fibrotic, inflammatory and growth pathways that lead to diastolic dysfunction in female mice.

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...

Shkryl VM, Maxwell JT, Domeier TL, Blatter LA. Refractoriness of sarcoplasmic reticulum Ca 2ϩ release determines Ca 2ϩ alternans in atrial myocytes.