Impairment of the adrenergic reserve associated with exercise intolerance in a murine model of heart failure with preserved ejection fraction.

AIM: Exercise intolerance is the central symptom in patients with heart failure with preserved ejection fraction. In the present study, we investigated the adrenergic reserve both in vivo and in cardiomyocytes of a murine cardiometabolic HFpEF model. METHODS: 12-week-old male C57BL/6J mice were fed regular chow (control) or a high-fat diet and L-NAME (HFpEF) for 15 weeks. At 27 weeks, we performed (stress) echocardiography and exercise testing and measured the adrenergic reserve and its modulation by nitric oxide and reactive oxygen species in left ventricular cardiomyocytes. RESULTS: HFpEF mice (preserved left ventricular ejection fraction, increased E/e', pulmonary congestion [wet lung weight/TL]) exhibited reduced exercise capacity and a reduction of stroke volume and cardiac output with adrenergic stress. In ventricular cardiomyocytes isolated from HFpEF mice, sarcomere shortening had a higher amplitude and faster relaxation compared to control animals. Increased shortening was caused by a shift of myofilament calcium sensitivity. With addition of isoproterenol, there were no differences in sarcomere function between HFpEF and control mice. This resulted in a reduced inotropic and lusitropic reserve in HFpEF cardiomyocytes. Preincubation with inhibitors of nitric oxide synthases or glutathione partially restored the adrenergic reserve in cardiomyocytes in HFpEF. CONCLUSION: In this murine HFpEF model, the cardiac output reserve on adrenergic stimulation is impaired. In ventricular cardiomyocytes, we found a congruent loss of the adrenergic inotropic and lusitropic reserve. This was caused by increased contractility and faster relaxation at rest, partially mediated by nitro-oxidative signaling.

Fluorescence Spectroscopy of Low-Level Endogenous ß-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes.

The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the ß1- and ß2-adrenergic receptors (ß1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of ß-ARs in adult CMs.

Visualization of ß-adrenergic receptor dynamics and differential localization in cardiomyocytes.

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope.

We report here on the measurement of the diffusion coefficient of fluorescent species using a commercial microscope possessing a resonant scanner. Sequential linescans with a rate of up to 12 kHz yield a temporal resolution of 83 µs, making the setup amenable to measure diffusion rates over a range covering at least three orders of magnitude, from 100 µm2/s down to 0.1 µm2/s. We share representative data sets covering (i) the diffusion of a dye molecule, observed in media of different viscosities and (ii) the diffusion of a prototypical membrane receptor. The data can be valuable for researchers interested in the rapid diffusion properties of nuclear, cytosolic or membrane bound proteins fused to fluorescent tags.