cGMP-Elevating Compounds and Ischemic Conditioning Provide Cardioprotection Against Ischemia and Reperfusion Injury via Cardiomyocyte-Specific BK Channels.

BACKGROUND: The nitric oxide-sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca2+-activated K+ channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types. METHODS: Gene-targeted mice with a cardiomyocyte- or smooth muscle cell-specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model. RESULTS: Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide-sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice. CONCLUSIONS: Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

cGMP Signaling in the Cardiovascular System—The Role of Compartmentation and Its Live Cell Imaging

The ubiquitous second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) regulates multiple physiologic processes in the cardiovascular system. Its intracellular effects are mediated by stringently controlled subcellular microdomains. In this review, we will illustrate the current techniques available for real-time cGMP measurements with a specific focus on live cell imaging methods. We will also discuss currently accepted and emerging mechanisms of cGMP compartmentation in the cardiovascular system

Role of Phosphodiesterase 1 in the Regulation of Real-Time cGMP Levels and Contractility in Adult Mouse Cardiomyocytes

In mouse cardiomyocytes, the expression of two subfamilies of the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase 1 (PDE1)—PDE1A and PDE1C—has been reported. PDE1C was found to be the major subfamily in the human heart. It is a dual substrate PDE and can hydrolyze both 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Previously, it has been reported that the PDE1 inhibitor ITI-214 shows positive inotropic effects in heart failure patients which were largely attributed to the cAMP-dependent protein kinase (PKA) signaling. However, the role of PDE1 in the regulation of cardiac cGMP has not been directly addressed. Here, we studied the effect of PDE1 inhibition on cGMP levels in adult mouse ventricular cardiomyocytes using a highly sensitive fluorescent biosensor based on Förster resonance energy transfer (FRET). Live-cell imaging in paced and resting cardiomyocytes showed an increase in cGMP after PDE1 inhibition with ITI-214. Furthermore, PDE1 inhibition and PDE1A knockdown amplified the cGMP-FRET responses to the nitric oxide (NO)-donor sodium nitroprusside (SNP) but not to the C-type natriuretic peptide (CNP), indicating a specific role of PDE1 in the regulation of the NO-sensitive guanylyl cyclase (NO-GC)-regulated cGMP microdomain. ITI-214, in combination with CNP or SNP, showed a positive lusitropic effect, improving the relaxation of isolated myocytes. Immunoblot analysis revealed increased phospholamban (PLN) phosphorylation at Ser-16 in cells treated with a combination of SNP and PDE1 inhibitor but not with SNP alone. Our findings reveal a previously unreported role of PDE1 in the regulation of the NO-GC/cGMP microdomain and mouse ventricular myocyte contractility. Since PDE1 serves as a cGMP degrading PDE in cardiomyocytes and has the highest hydrolytic activities, it can be expected that PDE1 inhibition might be beneficial in combination with cGMP-elevating drugs for the treatment of cardiac diseases

Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET.

Calcium (Ca2+) and 3',5'-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under β-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation

Ca²⁺ transient amplitudes in ISO and forskolin treated cardiomyocytes.

<p>Cells were loaded with Fura2-AM, paced at 1 Hz and treated with 100 nM ISO or 10 μM forskolin with subsequent applications of PDE inhibitors rolipram 10 μM, 8-MMX 30 μM and IBMX 100 μM. Shown are baseline Ca²⁺ amplitudes (black bars) and systolic Ca²⁺ transient amplitudes (grey bars) measured in paced <b>(A)</b> and resting <b>(B)</b> cells. Means ± SEM, *—p<0.05; **—p<0.01; ***—p<0.001 with ANOVA (compared to control, second value after / as compared to previous stimulation) followed by the Gasser-Greenhouse correction. n = 7 for ISO and n = 9 for forskolin cells in A, and n = 4 and 5 for ISO and forskolin cells in B, respectively (all isolated from at least 3 mice for each condition). Effects of ISO and forskolin in A are significantly different (p = 0.03 by one-way ANOVA).</p

Schematic diagram highlighting Ca²⁺ and cAMP changes observed in this study under different experimental conditions (basal, ISO and forskolin stimulated cardiomyocytes with and without pacing).

<p>Pacing leads to an increase in Ca²⁺ levels which is further augmented by forskolin>ISO via PKA-dependent phosphorylation of Ca²⁺ handling proteins. However, pacing has little effect on cAMP levels, apart from the case when it is combined with forskolin stimulation, together both lead to PDE1 activation. Increase of PDE1 activity affects presumably a discrete subcellular microdomain which constitutes a small percentage of the whole cellular AMP content and can be therefore revealed in the cytosol only by the use of a PDE1 inhibitor. Forskolin and ISO generate quantitatively comparable but differently shaped amounts of cAMP which may come from ISO-induced dissociation of PDE4D8 from the β₁-adrenergic receptor. This mechanism regulates local second messenger pool at the receptor and allows more rapid increase of cAMP in the cytosol, as compared to forskolin stimulation.</p

cAMP dynamics in adult mouse cardiomyocytes upon treatment with cAMP elevating agents and PDE1 inhibition.

<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or with the direct AC activator forskolin (10 μM) <b>(C)</b>. Subsequent application of the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX, 30 μM) enhances the cAMP stimulatory effect of ISO and forskolin. Stimulation with the unselective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX, 100 μM) leads to a further increase of cAMP. <b>(B and D)</b> Quantification of experiments shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with ISO. Forskolin stimulated cardiomyocytes show significant differences in PDE1 contribution to total PDE inhibition which is significantly higher in paced cardiomyocytes as compared to resting cells. Pretreatment of resting cardiomyocytes with calcium elevating reagents such as thapsigargin (100 nM) and calcium ionophore A23187 (10 μM) mimics the effect of field stimulation. Cells were paced at 1 Hz. Values are means ± SEM; n = 6–10 cells isolated from 3 hearts per condition; *—significant difference at p<0.05 by one-way ANOVA; n.s.- not significant.</p

cAMP dynamics in adult mouse ventricular Epac1-camps expressing cardiomyocytes upon treatment with cAMP elevating agents and PDE4 inhibition.

<p><b>(A)</b> Representative calcium traces in Fura2-AM loaded Epac1-camps transgenic cardiomyocytes under resting conditions (left) and upon electric field stimulation at 1 Hz (right). <b>(B)</b> Non-normalized FRET ratios do not differ between resting and paced Epac1-camps cardiomyocytes under basal and stimulated conditions (isoproterenol—ISO, 100 nM—plus 3-isobutyl-1-methylxathin—IBMX, 100 μM). <b>(C)</b> Representative FRET traces from Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or <b>(E)</b> with the direct AC activator forskolin (10 μM) leading to an increase of cAMP visualized as a decrease in the FRET ratio. Inhibition of PDE4 by rolipram (Roli, 10 μM) strongly enhances this effect, whereas the unselective PDE inhibitor IBMX (100 μM) has only little additional effect. <b>(D and F)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes. Cells were paced at 1 Hz. Values are means ± SEM; from n = 6 cells isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p