FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10–15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes.

Rationale: Cardiotoxic β1 adrenergic receptor (β1AR)-CaMKII signaling is a major and critical feature associated with development of heart failure. Synapse-associated protein 97 (SAP97) is a multi-functional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome. Objective: We aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure. Methods and Results: The integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac specific deletion of SAP97 was developed to examine β1AR signaling in ageing mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac specific deletion of SAP97 yields an ageing-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts. Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy.

Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of G i protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP 3 . By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP 3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP 3 -dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP 3 that leads to pronounced alterations of cytosolic cAMP levels.-Reddy, G. R., Subramanian, H., Birk, A., Milde, M., Nikolaev, V. O., Bünemann, M. Adenylyl cyclases 5 and 6 underlie PIP 3 -dependent regulation. FASEB J. 29, 3458-3471 (2015). www.fasebj.org