Airway diseases such as asthma are triggered by inflammation and mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα). Our goal was to systematically examine the potential mechanisms underlying the effect of TNFα on airway smooth muscle (ASM) contractility. Porcine ASM strips were incubated for 24 h with and without TNFα. Exposure to TNFα increased maximum ASM force in response to acetylcholine (Ach), with an increase in ACh sensitivity (hyperreactivity), as reflected by a leftward shift in the dose–response curve (EC50). At the EC50, the [Ca2+]cyt response to ACh was similar between TNFα and control ASM, while force increased; thus, Ca2+ sensitivity appeared to increase. Exposure to TNFα increased the basal level of regulatory myosin light chain (rMLC) phosphorylation in ASM; however, the ACh‐dependent increase in rMLC phosphorylation was blunted by TNFα with no difference in the extent of rMLC phosphorylation at the EC50 ACh concentration. In TNFα‐treated ASM, total actin and myosin heavy chain concentrations increased. TNFα exposure also enhanced the ACh‐dependent polymerization of G‐ to F‐actin. The results of this study confirm TNFα‐induced hyperreactivity to ACh in porcine ASM. We conclude that the TNFα‐induced increase in ASM force, cannot be attributed to an enhanced [Ca2+]cyt response or to an increase in rMLC phosphorylation. Instead, TNFα increases Ca2+ sensitivity of ASM force generation due to increased contractile protein content (greater number of contractile units) and enhanced cytoskeletal remodeling (actin polymerization) resulting in increased tethering of contractile elements to the cortical cytoskeleton and force translation to the extracellular matrix.

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sarcoplasmic reticulum (SR). A separate group of cardiomyocytes were fixed and key proteins were labelled with antibodies for subsequent confocal imaging. MCT myocytes had the largest cross-sectional area (4338 5 292mm 2) versus CON (3170 5 262mm 2 , P=0.01), although no difference in cell shortening was observed in response to 1 Hz stimulation. MCT myocytes had larger Ca 2þ transients at all stimulation frequencies (P= 0.004) and increased spontaneous activity during ß-adrenergic stimulation. No difference between groups was found in Ca 2þ store content, although the time constant of caffeine transient decay was prolonged in MCT (17.3 5 1.5s) versus CON (13.7 5 0.6s, P=0.05). Although there was no difference in shortening or SR Ca 2þ content, there was evidence of changes in Ca 2þ handling in MCT myocytes during inotropic stimulation. Furthermore, hypertrophic MCT myocytes exhibited slower transsarcolemmal Ca 2þ removal during 20mM caffeine, potentially as a result of disorganized T-tubular arrangement and/or reduced Na þ /Ca 2þ exchanger abundance.

The tongue can distinguish between five different tastes via the taste receptors, which are G-protein coupled receptors (GPCRs). There are two classes of taste receptors, the TAS1 (T1) and TAS2 (T2) families, and the T1R1-T1R3 dimer senses the umami taste and T1R2-T1R3 senses the sweet taste. Recently, the taste receptors have also been found in the brain, lungs, intestine and pancreas, where they sense changes in the nutrient environment and respond through GPCR signalling. Given the importance of glucose and amino acid metabolism in the heart, we hypothesized that the sweet and umami taste receptors have an important function in the heart. Using a variety of technologies and disease states, we have identified that T1R1, T1R2 and T1R3 are expressed in the heart. More specifically, mass spectrometry of a dog model of dyssynchrony has shown the presence of T1R1, T1R3 and T1R2. RNA seq of human patients who received a Left Ventricular Assist device and those who did not also revealed the presence of T1R1 and T1R3. The expression of these proteins was also confirmed using Western blot. We further showed T1R2 and T1R3 protein is localized in the plasma membrane of the cardiomyocytes by immunofluorescence (colocalized with Na/K ATPase) and PM enrichment. When we compared the taste receptor protein levels in dilated cardiomyopathy (DCM) compared to donor heart tissue, we found that T1R2 was overexpressed in DCM, showing that taste receptors may be important in nutrient sensing in disease. Furthermore, when neonatal rat ventricular myocytes were treated with sweet and umami agonists (aspartame for the sweet taste receptor and monosodium glutamate for the umami receptor), they had increased calcium transients as shown by an increase in peak calcium. Cardiomyocytes treated with aspartame also showed a decrease in time to relax. We hypothesize that in the heart, sweet and umami receptors induce positive inotropy upon a change in nutrient environment.

Ischemia/reperfusion (I/R) injury occurs during acute myocardial infarction and is a frequent cause of contractile failure, morbidity and mortality. There is evidence that reduced cardiac contractility following I/R injury is associated with disruption of excitation‐contraction (E‐C) coupling. We hypothesized that I/R injury alters E‐C coupling in cardiomyocytes by reducing the Ca2+ sensitivity of contraction.Cardiomyocytes were isolated from adult rat (Sprague Dawley) hearts using a modified Langendorff system. Cardiomyocytes were then loaded with fura‐2 AM (Ca2+ fluorescent indicator; 1 μM) and electrically stimulated every 2 s (0.5 Hz). Cytosolic Ca2+ concentration ([Ca2+]cyt) and sarcomere length shortening responses to stimulation were simultaneously measured using an IonOptix System equipped with a hypoxia‐regulated incubator system (Live Cell Instrument). To model I/R, cardiomyocytes were initially exposed to normoxia (21% O2) for 30 min followed by 30 min exposure to 2.5% O2 and finally 30 min exposure to normoxia. Throughout the I/R protocol, Ca2+ sensitivity of cardiomyocytes was assessed using a phase loop plot of concurrently measured [Ca2+]cyt and sarcomere length shortening.I/R reduced the extent of sarcomere length shortening, but it had no effect on the amplitude of evoked [Ca2+]cyt transients. Furthermore, I/R reduced Ca2+ sensitivity reflected by a rightward shift in the phase loop plots. These results support the hypothesis that I/R alters E‐C coupling in cardiomyocytes by reducing the Ca2+ sensitivity of contraction.Support or Funding InformationSupported by Mayo FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.