Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II‐induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+‐responsive transcriptional factor. It is believed that phospholipase C (PLC)‐mediated production of inositol‐1,4,5‐trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC‐mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II‐induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG‐sensitive TRPC channels, which leads to activation of L‐type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG‐sensitive TRPC‐like currents. Among DAG‐activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II‐induced NFAT activation and hypertrophic responses. These data suggest that DAG‐induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II‐induced NFAT activation and cardiac hypertrophy.

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric G_i/o proteins, resulting in the receptors being uncoupled from the G_i/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gα_i/o proteins by the A-protomer (G_i/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (G_i/o protein-independent action).

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)‐β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα12/13) in cardiomyocytes suppressed pressure overload‐induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF‐β, connective tissue growth factor, and periostin) and Ang‐converting enzyme (ACE) was suppressed by the functional inhibition of Gα12/13. The expression of these fibrogenic genes through Gα12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G‐protein‐coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα12/13 in cardiomyocytes by the extracellular nucleotides‐stimulated P2Y6 receptor triggers fibrosis in pressure overload‐induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF‐β.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH₂-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Gα_12/13-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Gα_12/13, but not by an RGS domain specific for Gα_q. Constitutively active Gα₁₂- or Gα₁₃-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Gα₁₃, which was completely inhibited by the Gα_12/13-specific RGS domain. Furthermore, the Gα_12/13-specific but not the Gα_q-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Gα_12/13-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Gα_12/13-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

Activation of Ca²⁺ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca²⁺ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca²⁺ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca²⁺ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca²⁺ influx itself or molecule(s) upstream of Ca²⁺ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr⁶⁹ and prevented TRPC6-mediated Ca²⁺ influx. Substitution of Ala for Thr⁶⁹ in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce Gα_q-mediated signaling, did not affect the suppression of receptor-activated Ca²⁺ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.