Murine cardiac growth, TRPC channels, and cGMP kinase I.

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.

Phospholipase D1 is involved in α1-adrenergic contraction of murine vascular smooth muscle.

α1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the α1-adrenergic-mediated signaling in vascular smooth muscle. α1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the α1-adrenergic-induced contraction and the α1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1(-/-) mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2(-/-) mice) but still present in the heterozygous control mice. α1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the α1-adrenergic-induced CaV1.2 channel activity.

The role of cGMP/cGKI signalling and Trpc channels in regulation of vascular tone.

AIMS: Signalling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (Trpc) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signalling. We tested this concept by investigating the role of cGMP/cGKI signalling on vascular tone and peripheral resistance using Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), Trpc1(-/-)/3(-/-)/6(-/-), and SM-specific cGKI(-/-) (sm-cGKI(-/-)) mice. METHODS AND RESULTS: α-Adrenergic stimulation induced similar contractions in L-NG-nitroarginine methyl ester (l-NAME)-treated aorta and comparably increased peripheral pressure in hind limbs from all mouse lines investigated. After α-adrenergic stimulation, 8-Br-cGMP diminished similarly aortic tone and peripheral pressure in control, Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice but not in sm-cGKI(-/-) mice. In untreated aorta, α-adrenergic stimulation induced similar contractions in the aorta from control and Trpc3(-/-) mice but larger contractions in sm-cGKI(-/-), Trpc6(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice, indicating a functional link between cGKI and Trpc6 channels. Trpc3 channels were detected by immunocytochemistry in both isolated aortic smooth muscle cells (SMCs) and aortic endothelial cells (ECs), whereas Trpc6 channels were detected only in ECs. Phenylephrine-stimulated Ca(2+) levels were similar in SMCs from control (Ctr) and Trpc6(-/-) mice. Carbachol-stimulated Ca(2+) levels were reduced in ECs from Trpc6(-/-) mice. Stimulated Ca(2+) levels were lowered by 8-Br-cGMP in Ctr but not in Trpc6(-/-) ECs. CONCLUSIONS: The results suggest that cGKI and Trpc1,3,6 channels are not functionally coupled in vascular SM. Deletion of Trpc6 channels impaired endothelial cGKI signalling and vasodilator tone in the aorta.

Protection through postconditioning or a mitochondria-targeted S-nitrosothiol is unaffected by cardiomyocyte-selective ablation of protein kinase G.

Protein kinase G type I (PKGI) plays a critical role in survival signaling of pre- and postconditioning downstream of cardiac cGMP. However, it is unclear whether PKGI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) metabolism can target cardiomyocyte mitochondria independently of cGMP/PKGI. We tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation and inhibition, adenosine A(2B) receptor (A(2B)AR) agonist, phosphodiesterase type-5 (PDE-5) inhibitor, or mitochondria-targeted S-nitrosothiol (MitoSNO) was affected by a cardiomyocyte-specific ablation of the PKGI gene in the mouse (CMG-KO). In situ hearts underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in CMG-CTRs all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10-s reperfusion and 10-s coronary occlusion) with or without treatment with the sGC inhibitor ODQ, treatment with the specific sGC activator BAY58-2667 (BAY58), the selective A(2B)AR agonist BAY60-6583 (BAY60), PDE-5 inhibitor sildenafil, and MitoSNO. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO· and S-nitrosates thiol proteins. In contrast, the hearts of CMG-KO animals were not protected by BAY58 and sildenafil, whereas the protective effects of IPost, IPost with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI is important for the protection against ischemia reperfusion injury afforded by sGC activation or PDE-5 inhibition. However, the beneficial effects of IPost, activation of the A(2B)AR, as well as the direct effects via mitochondrial S-nitrosation do not depend on PKGI in cardiomyocytes.

Cardiac hypertrophy is not amplified by deletion of cGMP-dependent protein kinase I in cardiomyocytes.

It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIbeta rescue mice (betaRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or betaRM but raised the heart weight by approximately 20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and betaRM and in isolated adult CMs cultured with or without 1 muM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and betaRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.