Abnormal β-adrenergic signaling plays a central role in human heart failure. In mice, chronic β-adrenergic receptor (βAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express βAR; however, the functional in vivo requirement of βAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. β2AR null mice exhibited attenuated hypertrophic responses to chronic βAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that β2AR activation in the heart induces pro-hypertrophic effects in mice. Since β2AR signaling is protective in cardiomyocytes, we focused on β2AR signaling in cardiac myofibroblasts. To determine whether β2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, β2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. β2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr). Furthermore, adenoviral overexpression of LTCC β-subunit (β) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that β phosphorylation occurs specifically in caveolae. Finally, β phosphorylation was observed after phenylephrine stimulation in β-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of β-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β dominantly localizes to caveolae and induces cardiac hypertrophy after α-adrenergic stimulation in mice. While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α-adrenergic stimulation using an originally developed tool.

Our study revealed that a single-dose injection of PLN-targeting LNA-ASO improved contractility in pressure overload-induced cardiac dysfunction, suggesting that LNA-ASO is a promising tool for hypertensive HF treatment.

Background and Objective: Inflammation occurs in the damaged heart and β adrenergic receptor (βAR) signaling could play an important role in cardiac inflammation. Cardiac fibroblasts (CFs) are one of the cardiac composing cells, and various stimuli including βAR activation promote cytokine production in CFs. Although CFs are involved in the development of heart failure via cytokine production, its molecular mechanism remains unclear. Therefore, we investigated to determine how βAR stimulation induces proinflammatory cytokines in CFs and to identify the new therapeutic targets preventing cardiac inflammation. Methods and Results: CFs were isolated from adult wild-type mice and the expression of proinflammatory cytokines, namely, IL-6, IL-1β and TNFα, was assessed by quantitative RT-PCR after βAR stimulation with isoproterenol (ISO, 1 or 10μM). As a result, IL-6 mRNA was upregulated 13-fold compared to basal condition 1 hour after ISO treatment. However, mRNA expression of IL-1β and TNFα showed little change after ISO treatment. Forskolin, in response to βAR-independent adenyl cyclase activator, the mRNA expression of IL-6, but not that of IL-1β or TNFα, was increased in a dose-dependent manner. Pretreatment with H-89, 5Z-7-Oxozeaenol or BAY11-7082 as PKA, TAK1 or NF-κB inhibitor, respectively, suppressed ISO-induced IL-6 upregulation. In contrast, N-acetylcysteine, an anti-oxidant, failed to reduce IL-6 mRNA increase. Furthermore, immunoblot analyses revealed that IL-1β-mediated induction of IL-6 was associated with activation of p-38 MAPK and JNK, while ISO did not activate these signaling pathway. Interestingly, the mRNA expression of Arid5a, an IL-6 mRNA stabilizer, showed 4-fold increase after 1 hour ISO treatment and suppressed its upregulation by the pretreatment with H-89, 5Z-7-Oxozeaenol or BAY11-7082. Finally, shRNA suppressing Arid5a expression decreased IL-6 mRNA induced by ISO treatment. Conclusion: In response toβAR stimulation, CFs produced IL-6 through Arid5a mainly by activating adenyl cyclase, PKA, TAK1 and NF-κB as a novel proinflammatory response in the heart.

Backgrounds: Recent studies demonstrated that the osteopontin (OPN), an acid phosphoprotein plays pivotal roles in cardiac hypertrophy and failure. An osteogenic transcription factor Runx2 regulates the expression of OPN in osteoblasts. In the present study, we examined the pathological role of Runx2 in cardiac hypertrophy and failure. Methods and Results: Runx2 expression was detected in neonatal cardiomyocytes and upregulated in heart 14 days after myocardial infarction (MI) as well as 7days after transverse aortic constriction (TAC) procedures. To determine the functional role of Runx2 in heart, we generated transgenic mice (TG) with inducible cardiac-specific overexpression of Runx2. Two TG lines (low and high) were obtained and high-expressing TG (HE-TG) showed premature death within 8 weeks of age specifically in male mice. At two months of age, the survived male and female HE-TG displayed significant increases in heart weight/body weight ratio (mg/g) compared to controls (control; 4.95±0.26, n=6 vs HE-TG; 6.63±0.12, n=5, p<.05). Consistent with those results, the expression of hypertrophic marker genes such as atrial natriuretic factor (ANF) and αskeletal actin significantly increased in HE-TG heart assessed by real-time RT-PCR analysis. In addition, HE-TG mice demonstrated decreased fractional shortening assessed by echocardiography (control; 44.1±1.89%, n=9 vs HE-TG; 23.9±3.48%, n=7, p<.05). HE-TG mice demonstrated significantly lower heart rate (control; 630±18 bpm, vs HE-TG; 350±74 bpm, n=3 each, p<.05) and complete atrioventricular block by telemetry analysis. In response to pressure overload, low expressing TG (LE-TG) demonstrated higher mortality and enhanced cardiac hypertrophic response after TAC (control; 6.20±0.23, n=6 vs LE-TG; 6.90±0.26, n=4, p<.05). Conclusions: Targeted expression of Runx2 in heart mediates cardiac dysfunction and hypertrophy in mice. Thus, Runx2 could be a novel therapeutic target for heart failure.