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. To this end, we investigated the role of such biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM) comprising collagen type I and neonatal rat heart cells. Following tissue consolidation (8 days), 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, increased amounts of SERCA2a and RyR2 protein, and enhanced T-tubulation. We demonstrate that electromechanical stimulation at a frequency matching closely the physiological heart rate supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research and drug testing.

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 β-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated β-adrenoceptormediated 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, Ca 2+ -spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca 2+ leakage and lower basal phosphorylation levels of Ca 2+ -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. (Circ Res. 2017;120:120-132. DOI: 10.1161/ CIRCRESAHA.116.310069.) Key Words: cardiac arrhythmia ■ catecholamine ■ cyclic GMP-stimulated phosphodiesterase ■ heart rate ■ myocardial contractionOriginal received October 2, 2016; revision received October 27, 2016; accepted October 31, 2016. In September 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.73 days.From the Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Mannheim, Heidelberg University, Germany (C.V., T.W.); Institute of Pharmacology, University Medical Center Göttingen (UMG) Heart Center, Georg August University Medical School Göttingen, Germany (C.V., M.D., M.R., S.M.); UMR-S 1180, INSERM, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France (M.L., H.M., S.K., P.L., J.L., G.V., R.F.); Department of Molecular Cardiology and Epigenetics, University Hospital Heidelberg, Germany (M.D.); Institute of Pharmacology and Toxicology, University of Würzburg and Leibniz-Institut für Analytische Wissenschaften -ISAS -e.V., Dortmund, Germany (K.L., ...