Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.

Secondhand smoke exposure impairs ion channel function and contractility of mesenteric arteries

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 hours/day, 5 days/week) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 weeks but not for 4 or 8 weeks. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 weeks of SHS exposure. Moreover, 12 weeks SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

Abstract 12677: Impaired Cardiomyocyte Contractility to Angiotensin-(1-12) in a Humanized Angiotensinogen Model of Hypertension

Background: Angiotensin-(1-12) [Ang-(1-12)] is a chymase-dependent source for Angiotensin II (Ang II) inotropic activity that may be impaired in a model of sustained hypertension with high cardiac Ang II content due to insertion of the human angiotensinogen (AGT) gene in the rat genome. Accordingly, we evaluated the effects of Ang-(1-12) and Ang II on myocyte contractility and [Ca 2+ ] i regulation in 9 adult male transgenic rats expressing the human AGT gene [TGR(hAGT)L1623)] and 9 SD controls. Methods: We compared LV myocyte contraction, relaxation and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to Ang-(1-12) (4x10 -6 M) and Ang II (10 -6 M) in freshly isolated LV myocytes. Results: Myocyte contraction (dL/dt max , 109.6 vs 127.9 μm/s), relaxation (dR/dt max , 95.3 vs 107.5 μm/s) and [Ca 2+ ] iT (0.15 vs 0.24) were depressed in TGR(hAGT)L1623 rats compared to SD controls. Moreover, cell contractile and [Ca 2+ ] iT responses following exposure to Ang-(1-12) or Ang II were markedly blunted. In SD myocytes, versus baseline, Ang II or Ang-(1-12) superfusion produced significant increases in dL/dt max [Ang II: 44% vs Ang-(1-12): 34%], dR/dt max (33% vs 26%) and [Ca 2+ ] iT (31% vs 25%). Importantly, the magnitude of the responses to the two agents in TGR(hAGT)L1623 myocytes was significantly reduced. Versus the changes in SD myocytes, Ang-(1-12) caused significantly less increases in dL/dt max (22%), dR/dt max (16%) and [Ca 2+ ] iT (15%) in TGR(hAGT)L1623 myocytes . Ang II also caused similar significantly attenuated increases in dL/dt max (27%), dR/dt max (25%) and [Ca 2+ ] iT (23%). The Ang-(1-12)-induced inotropic effects were completely prevented in the presence of the inhibitory cAMP analog, Rp-cAMPS (10 –4 M, 2 hours) in both SD and TGR(hAGT)L1623 myocytes, but were further augmented only intransgenic rats after incubation of myocytes with the G i inhibitor, pertussis toxin (PTX, 2 μg/ml, 36°C, 5 hours). Conclusions: Ang-(1-12) stimulates LV myocyte contractile function and [Ca 2+ ] iT in both SD and TGR(hAGT)L1623 rats. Furthermore, we now show that the blunted inotropic responses to Ang-(1-12) and Ang II in rats expressing the human AGT gene is mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Abstract 13045: Chronic Beta3-Adrenergic Receptor Antagonist Therapy Rescues Diabetic Cardiomyopathy in a Mouse Model With Type 2 Diabetes: Insights into Cellular Mechanism

Background: Diabetic cardiomyopathy (DCM) leads to progressive decline in cardiac function, increasing the risk for heart failure. There are no known effective prevention approaches or therapeutic strategies. Recent evidence in diabetes mellitus (DM) human and animal hearts suggests that the up-regulation of β 3 -adrenergic receptor (AR)-mediated inhibitory pathway may be responsible for the progression of DCM. However, its precise role is still being debated. We hypothesize that β 3 -AR antagonists (β 3 -ANT) may rescue the detrimental effects of β 3 -AR activation, improve cardiomyocyte function, and preserve normal β-AR regulation, leading to the regression of DCM. Methods: We compared LV myocyte function, [Ca 2+ ] i transient ([Ca 2+ ] iT ) at baseline and responses to β-AR simulation with isoproterenol (ISO,10 -8 M) in 3 groups wild-type female mice over 14 weeks (W):1) Type 2 DM ( T2 ) (n=9), 14 W fed high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); 2) T2/β 3 -ANT (n=7), T2 mice at 10 W received L-748,337, a selective β 3 -ANT (10 -7 M/kg/day, mini-pump) for 4 W; and 3) Vehicle controls (C) (n=9). Results: Versus C, T2DM was induced in mice received HFD and low dose STZ with significantly elevated blood glucose levels (T2: 388, T2/β 3 -ANT: 369 vs C: 128 mg/dl). In T2, LV myocyte basal function and [Ca 2+ ] iT regulation were impaired measured as significantly decreased myocyte contractility (dL/dt max ) (76.8 vs 135.2 μm/s), relengthening (dR/dt max ) (62.1 vs 113.8 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). Furthermore, versus C, in T2 myocytes, ISO-induced increases in dL/dt max (T2: 40% vs C: 58%), dR/dt max (35% vs 54%) and [Ca 2+ ] iT (19% vs 30%) were significantly reduced. By contrary, versus T2, T2/β 3 -ANT myocytes showed normal basal cell contraction (127.8 μm/s), relaxation (109.4 μm/s) and [Ca 2+ ] iT (0.21) with preserved ISO-stimulated positive inotropic effect. Versus C, in T2/β 3 -ANT, ISO caused similar increases in dL/dt max (57%), dR/dt max (52%) and [Ca 2+ ] iT (30%). Conclusion: Chronic β 3 -ANT leads to the preservation of LV myocyte function, [Ca 2+ ] iT , and β-AR responsiveness in a mouse model of type 2 diabetes. Thus, antagonizing β 3 -AR might provide a new therapeutic strategy for DM-related decline in myocardial function.

Abstract 12808: Mechanism of Chronic Ranolazine Caused Regression of Cardiac Dysfunction in a Murine of Diabetic Cardiomyopathy: Beneficial Effects on Cardiomyocyte Contractile Function, [Ca 2+ ] i Regulation and Beta-Adrenergic Modulation

Background: Diabetic cardiomyopathy (DCM) increases the risk of heart failure. As yet, no effective therapeutic strategies exist. Recent evidence indicates that intracellular Na + concentration ([Na + ] i ) is augmented in the myocytes from diabetic hearts, where it causes oxidative stress, augments the sarcoplasmic reticulum Ca 2+ leak and contributes to electrical, structural and functional remodeling. Ranolazine (RAN), inhibiting persistent or late inward Na + current has been proposed to be a therapeutic choice for DCM. However, the role and mechanism of chronic RAN in DCM are unclear. We assessed the hypothesis that RAN improves myocyte function, [Ca 2+ ] i regulation, and β-adrenergic receptor (AR) signaling effectiveness, thus limiting DCM. Methods: We compared LV myocyte function, [Ca 2+ ] i transient ([Ca 2+ ] iT ) and responses to the stimulation of β-AR in 3 groups wild-type (WT) female mice over 10 weeks (W):1) DM (n=8), 10 W after receiving streptozotocin (STZ, 200 mg/kg, ip); 2) DM/RAN (n=6), 6 W after STZ, RAN (10 -5 M/kg/day, mini-pump) was initiated and was given for 4 W; and 3) Sham controls (C) (n=8). Results: Versus control, STZ-treated WT mice had DM with significantly elevated blood glucose levels (410 vs 128mg/dl) followed by LV myocyte dysfunction with decreases in myocyte contractility (dL/dt max ) (75.0 vs 140.1 μm/s), relengthening (dR/dt max ) (62.5 vs 116.6 μm/s) and [Ca 2+ ] iT (0.15 vs 0.22). In DM myocytes, the ability of β-AR agonist, isoproterenol (ISO, 10 -8 M) to increase cell contractility was blunted. Versus control, in DM myocytes, ISO-induced increases in dL/dt max (31% vs 60%), dR/dt max (23% vs 50%) and [Ca 2+ ] iT (15% vs 30%) were significantly reduced. By contrary, versus DM alone, DM/RAN myocytes showed normal basal cell contraction (137.8 μm/s), relaxation (117.2 μm/s) and [Ca 2+ ] iT (0.22) with preserved ISO-stimulated positive inotropic effect. Compared control, in DM/RAN, ISO caused similar increases in dL/dt max (62% vs 60%), dR/dt max (52% vs 50%) and [Ca 2+ ] iT (32% vs 30%). Conclusion: Chronic ranolazine leads to the preservation of myocyte function, [Ca 2+ ] iT and β-AR responsiveness in DCM. Thus, antagonizing myocyte [Na + ] i dysregulation might provide a new therapeutic strategy for DM-related decline in myocardial function.

Regulating quantal size of neurotransmitter release through a GPCR voltage sensor

Current models emphasize that membrane voltage (Vm) depolarization-induced Ca2+ influx triggers the fusion of vesicles to the plasma membrane. In sympathetic adrenal chromaffin cells, activation of a variety of G protein coupled receptors (GPCRs) can inhibit quantal size (QS) through the direct interaction of G protein Giβγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca2+, Vm (action potential) per se regulates the amount of catecholamine released from each vesicle, the QS. The Vm regulation of QS was through ATP-activated GPCR-P2Y12 receptors. D76 and D127 in P2Y12 were the voltage-sensing sites. Finally, we revealed the relevance of the Vm dependence of QS for tuning autoinhibition and target cell functions. Together, membrane voltage per se increases the quantal size of dense-core vesicle release of catecholamine via Vm → P2Y12(D76/D127) → Giβγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs.

Abstract 416: Sarcomere Disassembly After Unloading is Regulated by Ubiquitination and Acetylation of Capz and α-actinin

Cardiac function mainly depends on the total myocyte mass in the ventricles. Assembly and disassembly of sarcomeres occurs to adjust this mass to altered mechanical demand. In the heart, hypertrophic cardiomyopathy results from myofibrillar assembly controlled by post-translational modification of proteins directed by signaling pathways. More is known about assembly on loading than disassembly on unloading. Here, the hypothesis tested is that unloading of mechanical forces affects acetylation (Ac) and ubiquitination (Ub) of the actin-binding proteins, α-actinin and CapZ. Omecamtiv mecarbil (0.5 μM) and mavacamten (1 μM) were used to increase (load) and decrease (unload) cardiomyocyte tension, respectively, via their action on myosin ATPase. Mavacamten decreased myocyte contractility in rat ventricular myocytes (NRVMs) and caused significant sarcomere disassembly by 6 h and 70% atrophy by 24 h. Assembly was preserved with omecamtiv mecarbil (0.5 μM) over the 24 h time period. Post-translational modification was determined in loaded and unloaded NRVMs at 6 h of drug treatment. Bottom-up mass spectrometry analysis showed single residues in α-actinin and CapZ that were acetylated or ubiquitinated. Acetylation levels appeared to increase in the mavacamten-treated samples while these levels are preserved in untreated and omecamtiv mecarbil-treated samples. Ac and Ub in the Z-discs were quantified on immunofluorescent images. The Z-discs colocalized oligo-Ub (K-48 oligo-Ub linkage) and Ac in untreated samples; this Z-disc localization of Ub and Ac was diminished with unloading. Fluorescence recovery after photobleaching (FRAP) measurements of the dynamics of α-actinin and CapZ after reduced cell tension with mavacamten (1 μM) and omecamtiv mercabil (0.5 μM) are ongoing. Overall, results suggest sarcomere assembly is regulated by mechanical forces through a mechanism involving Ac and Ub of myofibrillar proteins. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis.

TRPML1ng on sparks

In this issue of Science Signaling, Thakore et al. report that the Ca2+-permeable channel TRPML1 closely associates with ryanodine receptors to induce Ca2+ sparks in native arterial myocytes. Functional studies revealed a key role for TRPML1 channels in regulation of arterial myocyte contractility and blood pressure.