CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2.

BACKGROUND: Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways. METHODS: To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes. RESULTS: In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in ICaL, INaL, and Ca2+ spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca2+ handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion. CONCLUSIONS: CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.

Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction.

RATIONALE: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown. OBJECTIVE: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction. METHODS AND RESULTS: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal ß-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated ß-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction. CONCLUSIONS: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction.

Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, ß, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of ß-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1ß levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

The reduced activity of PP-1α under redox stress condition is a consequence of GSH-mediated transient disulfide formation.

Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to ß-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called "cross-over-read" search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.

Calcium/Calmodulin-Dependent Protein Kinase II Activity Persists During Chronic ß-Adrenoceptor Blockade in Experimental and Human Heart Failure.

BACKGROUND: Considerable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive ß-adrenoceptor (ß-AR) stimulation. Recent studies indicate a significant cross talk between ß-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental ß-AR signaling in HF. In this study, we investigated the effect of chronic ß-AR blocker treatment on CaMKII activity in human and experimental HF. METHODS AND RESULTS: Immunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with ß-AR blockers revealed no difference in CaMKII activity when compared with non-ß-AR blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδC transgenic mice were treated with the ß1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29; P<0.001), attenuated the development of cardiac hypertrophy (-14±6% heart weight/tibia length; P<0.05), and strongly reduced ventricular arrhythmias (-70±22% premature ventricular contractions; P<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A-dependent phospholamban and ryanodine receptor 2 phosphorylation (-42±9% for P-phospholamban-S16 and -22±7% for P-ryanodine receptor 2-S2808; P<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by ß-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol. CONCLUSIONS: Chronic ß-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of ß-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to ß-AR blockade.

Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation.

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. We investigated the role of biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM). Following tissue consolidation, EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum and enhanced T-tubulation. We conclude that electro-mechanical stimulation at a physiological frequency supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research.