Myocyte Contractility Research Articles

Ameliorating Effect of Klotho Protein on Rat Heart during I/R Injury.

An essential procedure for the treatment of myocardial infarction is restoration of blood flow in the obstructed infarct artery, which may cause ischaemia/reperfusion (I/R) injury. Heart I/R injury manifests in oxidative stress, metabolic and morphological disorders, or cardiac contractile dysfunction. Klotho protein was found to be produced in the heart tissue and participate in antioxidation or ion homeostasis. The aim of this study was to examine an influence of Klotho protein on the heart subjected to I/R injury. Wistar rats served as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischaemia, and isolated rat cardiomyocytes underwent chemical I/R in vitro, with or without recombinant Klotho protein administration. Haemodynamic parameters of heart function, cell contractility, markers of I/R injury and oxidative stress, and the level of contractile proteins such as myosin light chain 1 (MLC1) and troponin I (TnI) were measured. The treatment of hearts subjected to I/R injury with Klotho protein resulted in a recovery of heart mechanical function and ameliorated myocyte contractility. This improvement was associated with decreased tissue injury, enhanced antioxidant capacity, and reduced release of MLC1 and TnI. The present research showed the contribution of Klotho to cardioprevention during I/R. Thus, Klotho protein may support the protection from I/R injury and prevention of contractile dysfunction in the rat heart.

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.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium‐independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium‐transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura‐2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Influence of Crocetin, a Natural Carotenoid Dicarboxylic Acid in Saffron, on L-Type Ca2+ Current, Intracellular Ca2+ Handling and Contraction of Isolated Rat Cardiomyocytes.

Crocetin is a major bioactive ingredient in saffron (Crocus sativus L.) and has favorable cardiovascular effects. Here, the effects of crocetin on L-type Ca2+ current (ICa-L), contractility, and the Ca2+ transients of rat cardiomyocytes, were investigated via patch-clamp technique and the Ion Optix system. A 600 µg/mL dose of crocetin decreased ICa-L 31.50 ± 2.53% in normal myocytes and 35.56 ± 2.42% in ischemic myocytes, respectively. The current voltage nexus of the calcium current, the reversal of the calcium current, and the activation/deactivation of the calcium current was not changed. At 600 µg/mL, crocetin abated cell shortening by 28.6 ± 2.31%, with a decrease in the time to 50% of the peak and a decrease in the time to 50% of the baseline. At 600 µg/mL, crocetin abated the crest value of the ephemeral Ca2+ by 31.87 ± 2.57%. The time to half maximal of Ca2+ peak and the time constant of decay of Ca2+ transient were both reduced. Our results suggest that crocetin inhibits L-type Ca2+ channels, causing decreased intracellular Ca2+ concentration and contractility in adult rat ventricular myocytes. These findings reveal crocetin's potential use as a calcium channel antagonist for the treatment of cardiovascular disease.

Diazoxide preserves myocardial function in a swine model of hypothermic cardioplegic arrest and prolonged global ischemia

ObjectiveAdenosine triphosphate potassium sensitive channels provide endogenous myocardial protection via coupling of cell membrane potential to myocardial metabolism. Adenosine triphosphate potassium sensitive channel openers, such as diazoxide, mimic ischemic preconditioning, prevent cardiomyocyte swelling, preserve myocyte contractility after stress, and provide diastolic protection. We hypothesize that diazoxide combined with hyperkalemic cardioplegia provides superior myocardial protection compared with cardioplegia alone during prolonged global ischemia in a large animal model. MethodsTwelve pigs were randomized to global ischemia for 2 hours with a single dose of cold blood (4:1) hyperkalemic cardioplegia alone (n = 6) or with diazoxide (500 μmol/L) (n = 6) and reperfused for 1 hour. Cardiac output, myocardial oxygen consumption, left ventricular developed pressure, left ventricular ejection fraction, diastolic function, myocardial troponin, myoglobin, markers of apoptosis, and left ventricular infarct size were compared. ResultsFour pigs in the cardioplegia alone group could not be weaned from cardiopulmonary bypass. There were no differences in myoglobin, troponin, or apoptosis between groups. Diazoxide preserved cardiac output versus control (74.5 vs 18.4 mL/kg/min, P = .01). Linear mixed regression modeling demonstrated that the addition of diazoxide to cardioplegia preserved left ventricular developed pressure by 36% (95% confidence interval, 9.9-61.5; P < .01), dP/dt max by 41% (95% confidence interval, 14.5-67.5; P < .01), and dP/dt min by 33% (95% confidence interval, 8.9-57.5; P = .01). It was also associated with higher (but not significant) myocardial oxygen consumption (3.7 vs 1.4 mL O2/min, P = .12). ConclusionsDiazoxide preserves systolic and diastolic ventricular function in a large animal model of prolonged global myocardial ischemia. Diazoxide as an adjunct to hyperkalemic cardioplegia may allow safer prolonged ischemic times during increasingly complicated cardiac procedures.

Early changes in myocyte contractility and cardiac function in streptozotocin-induced type 1 diabetes in rats

Diabetes can elicit direct deleterious effects on the myocardium, independent of coronary artery disease or hypertension. These cardiac disturbances are termed diabetic cardiomyopathy showing increased risk of heart failure with or without reduced ejection fraction. Presently, there is no specific treatment for this type of cardiomyopathy and in the case of type I diabetes, it may start in early childhood independent of glycemic control. We hypothesized that alterations in isolated myocyte contractility and cardiac function are present in the early stages of experimental diabetes in rats before overt changes in myocardium structure occur. Diabetes was induced by single-dose injection of streptozotocin (STZ) in rats with data collected from control and diabetic animals 3 weeks after injection. Left ventricle myocyte contractility was measured by single-cell length variation under electrical stimulation. Cardiac function and morphology were studied by high-resolution echocardiography with pulsed-wave tissue Doppler imaging (TDI) measurements and three-lead surface electrocardiogram. Triglycerides, cholesterol and liver enzyme levels were measured from plasma samples obtained from both groups. Myocardial collagen content and perivascular fibrosis of atria and ventricle were studied by histological analysis after picrosirius red staining. Diabetes resulted in altered contractility of isolated cardiac myocytes with increased contraction and relaxation time intervals. Echocardiography showed left atrium dilation, increased end-diastolic LV and posterior wall thickness, with reduced longitudinal systolic peak velocity (S’) of the septum mitral annulus at the apical four-chamber view obtained by TDI. Triglycerides, aspartate aminotransferase and alkaline phosphatase were elevated in diabetic animals. Intertitial collagen content was higher in atria of both groups and did not differ among control and diabetic animals. Perivascular intramyocardial arterioles collagen did not differ between groups. These results suggest that alterations in cardiac function are present in the early phase in this model of diabetes type 1 and occur before overt changes in myocardium structure appear as evaluated by intersticial collagen deposition and perivascular fibrosis of intramyocardial arterioles.

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.

Electrical impedance-based contractile stress measurement of human iPSC-Cardiomyocytes

Heart failure fundamentally results from loss of cardio myocyte contractility. Developing new methods that quantify the contractile stress of the human cardiomyocyte would facilitate the study of the molecular mechanism of heart failure and advance therapy development, to improve the current five year survival for these patients. The measurement of cellular electrical impedance measurement was recently applied to monitor cardiomyocyte beating rate and rhythm, for the study at cellular maturation, and for drug screening. However, due to the lack of a quantified relationship between the impedance signal and contractile stress, change of cardiomyocyte contractile stress cannot genuinely be quantified from impedance measurements. Here, we report the first quantitative relationship between contractile stress and impedance, which enables the accurate prediction of cardiomyocyte contractility using impedance signals. Through simultaneous measurement of beating human iPSC-cardiomyocytes using impedance spectroscopy and atomic force microscopy, a power-law relationship between impedance and contractile stress was established with a confidence level of 95%. The quantitative relationship was validated using pharmacology known to alter cardiomyocyte contractility and beating (verapamil, using clinically relevant concentrations of 0.05 μM, 0.10 μM, and 0.15 μM). The contractile stress values as measured by AFM were 9.04 ± 0.14 kPa (0.05 μM), 7.72 ± 0.11 kPa (0.10 μM) and 6.23 ± 0.17 kPa (0.15 μM), and as predicted by impedance using the derived power-law relationship were 9.39 kPa, 7.76 kPa, and 6.05 kPa with a relative error of 3.73%. Our power-law relationship is the first to describe a quantitative correlation between contractile stress and impedance, broadening the application of electrical impedance measurement for characterizing complex cardiac functions (beating rate, beating rhythm and contractile stress).

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Towards understanding the role of Receptor Expression Enhancing Protein 5 (REEP5) in cardiac muscle and beyond.

The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.

SAT-498 Graves’-Associated Takotsubo Cardiomyopathy: An Uncommon Condition

Introduction Graves’ disease is an autoimmune disorder that causes excess thyroid hormone (T4 and T3). T4 is converted into T3 (active hormone) in the peripheral tissues by the deiodinase enzyme. T3 has an effect on the cardiac electrical system as well as myocyte contractility. Excess T3 can result in cardiac arrythmias as well as ventricular dysfunction (heart failure). Takotsubo cardiomyopathy is a subtype of nonischemic cardiomyopathy related to severe physiologic or mental stress. Excess catecholamine levels have been reported to cause this disease as well. Takotsubo cardiomyopathy has rarely been reported in Graves’-associated thyrotoxicosis. Case report 55-year-old female who presented to the ED with palpitations and difficulty breathing. She had been seen by her PCP within the past two weeks with complaint of recent 30 lb weight. TSH was noted to be < 0.001 and a neck ultrasound revealed a diffusely enlarged, hypervascular thyroid. She was referred for outpatient endocrinology consultation for further workup. Prior to her initial endocrinology appointment, she developed palpitations and shortness of breath. She did not have any known family history of thyroid disease or other autoimmune conditions. She drinks 1-2 glasses of wine per week and quit smoking in 2017. Physical exam on admission revealed a heart rate of 133 bpm and a blood pressure of 145/93, normal temperature, and normal respirations. Thyroid was diffusely enlarged and without nodularity. No evidence of orbitopathy. Heart was tachycardic but without murmur. Lungs clear. Abdomen soft, nontender. No significant peripheral edema or pretibial rash. No neurological dysfunction. Labs revealed TSH < 0.001, Free T4 > 7.77, Free T3 12.0, TRAb 44.4%, TSI 433%, Anti-TPO 614, high-sensitivity troponin of 112. EKG showed nonspecific infero-lateral T wave changes, rate 123. Echocardiogram demonstrated left ventricular apical hypokinesis but preserved basilar contractility. Ejection fraction was estimated at 40-45%. Nuclear stress test did not reveal any indication of myocardial hypoperfusion. Discussion/Conclusion Takotsubo cardiomyopathy can mimic acute myocardial infarction both clinically as well as on EKG/serum biomarkers. Troponin levels are typically elevated as a result of myocardial stretch and subsequent troponin “leak”. Echocardiogram demonstrates apical ballooning of the left ventricle, and by definition coronary arteries will be free of significant occlusive disease. A small number of cases have been reported in association with endocrine conditions including thyrotoxicosis due to Graves’ disease. The majority of cases associated with thyrotoxicosis will resolve spontaneously with 1-3 weeks. Treatment consists of medication to decrease cardiac preload as well as afterload (ACE inhibitor, beta blocker, diuresis as needed), similar to medical treatment of other nonischemic cardiomyopathies.

OCT3 gated intracellular β1AR activation enhances excitation‐contraction coupling in heart

Rationaleβ1AR‐adrenergic receptor (β1AR) plays a central role in sympathetic regulation of cardiac excitation‐contraction (EC) coupling. Stimulation of β1AR by catecholamines enhances cardiomyocyte contraction and Ca2+transient via the cAMP‐protein kinase A (PKA) pathway. Organic cation transporter 3 (OCT3) plays a role in cleaning up the extracellular catecholamines and transporting a portion of catecholamine into myocardium. Recent progress found that β1AR is also present at intracellular membranes. However, how the intracellular β1AR was activated, and the role of OCT3 in regulating intracellular β1AR signaling and EC‐coupling remains unknown.ObjectiveTo characterize the presence and activation of intracellular β1AR‐adrenergic receptors and their impact on cardiac EC‐couplingMethods and ResultsWe used a genetic deletion of OCT3 to inhibit the entrance of catecholamine into the cells. We used isolated myocytes to measure β1AR‐induced contractility and Ca2+transients. We used PKA activity reporters to detect local activation via intracellular β1AR. We found that catecholamine‐induced inotropic effects were diminished in OCT3‐knock out hearts. OCT3 deficiency significantly attenuated norepinephrine (NE) induced PKA activities at the sarcoplasmic reticulum, myocyte contractility, and Ca2+transient. Additional data show β1AR associates with sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) at the sarcoplasmic reticulum (SR). Inhibition of OCT3 blocked the transportation of NE, and attenuated β1AR‐induced PKA activity at the SR and EC‐coupling.ConclusionOur finding demonstrates a previously unrecognized intracellular β1AR‐mediated local PKA activity at SR that is gated by OCT3. Upon catecholamine stimulation, this SR‐localized β1AR signal plays a critical role in promoting E‐C coupling and cardiac contractile function. Thus, internal β1AR signaling may presents a novel therapeutic target in cardiac diseases treatment.Support or Funding InformationNIH HL113413VABX009200

Cardiac‐specific Deletion of Filamin C Decouples Myocyte Contractility and Calcium Handling in Adult Mice

Costameres are sub‐membranous protein complexes in striated muscle that play roles in mechanical signal transduction and force transmission between the Z‐disc of the sarcomere and the extracellular matrix. In cardiac myocytes (CMs), the cytoskeletal protein Filamin C (FLNC) interacts with multiple proteins in the Z‐disc and the costamere, suggesting that it contributes to costamere‐based mechanotransduction in the heart. Moreover, mutations in the gene that encodes FLNC have been associated with multiple forms of human cardiomyopathies. To investigate the role of FLNC in cardiac mechanosignaling and pathological remodeling, we employed a recently developed murine model of inducible and cardiac‐specific FLNC knockout (FLNC‐icKO). Previous studies with this mouse model used echocardiographic measurements of adult FLNC‐icKO mice to reveal a severe dilated cardiomyopathy (DCM) phenotype by 2 weeks post‐induction, including significantly reduced ventricular fractional shortening. Correspondingly, we found that intact papillary muscles dissected from adult FLNC‐icKO hearts have significantly reduced twitch force compared to those dissected from wild‐type (WT) hearts. Interestingly, while the fractional shortening of isolated CMs from FLNC‐icKO hearts is also significantly reduced compared to WT CMs, the calcium transient (peak amplitude and duration) during a twitch is not different between groups. These results suggest that contractility and calcium handling in FLNC‐null hearts are decoupled. Potential mechanisms that may be responsible for this apparent decoupling are a disruption in force transmission between the sarcomere and the cell membrane, an increased myofilament lattice spacing, or altered calcium sensitivity of the myofilaments upon the deletion of FLNC in CMs.Support or Funding InformationFunding: HL007444‐36 to JDP, HL144872 to JC, and HL137100 to ADM.

Impact of hyperthyroidism on cardiac hypertrophy.

The cardiac growth process (hypertrophy) is a crucial phenomenon conserved across a wide array of species and is critically involved in the maintenance of cardiac homeostasis. This process enables an organism to adapt to changes in systemic demand and occurs due to a plethora of responses, depending on the type of signal or stimuli received. The growth of cardiac muscle cells in response to environmental conditions depends on the type, strength and duration of stimuli, and results in adaptive physiological responses or non-adaptive pathological responses. Thyroid hormones (TH) have a direct effect on the heart and induce a cardiac hypertrophy phenotype, which may evolve to heart failure. In this review, we summarize the literature on TH function in the heart by presenting results from experimental studies. We discuss the mechanistic aspects of TH associated with cardiac myocyte hypertrophy, increased cardiac myocyte contractility and electrical remodeling, as well as the associated signaling pathways. In addition to classical crosstalk with the sympathetic nervous system (SNS), emerging work pointing to the new endocrine interaction between TH and the renin-angiotensin system (RAS) is also explored. Given the inflammatory potential of the angiotensin II peptide, this new interaction may open the door for new therapeutic approaches which target the key mechanisms responsible for TH-induced cardiac hypertrophy.

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

Potential of the Cardiovascular Drug Levosimendan in the Management of Amyotrophic Lateral Sclerosis: An Overview of a Working Hypothesis.

Levosimendan is a calcium sensitizer that promotes myocyte contractility through its calcium-dependent interaction with cardiac troponin C. Administered intravenously, it has been used for nearly 2 decades to treat acute and advanced heart failure and to support the heart function in various therapy settings characterized by low cardiac output. Effects of levosimendan on noncardiac muscle suggest a possible new application in the treatment of people with amyotrophic lateral sclerosis (ALS), a neuromuscular disorder characterized by progressive weakness, and eventual paralysis. Previous attempts to improve the muscle response in ALS patients and thereby maintain respiratory function and delay progression of disability have produced some mixed results. Continuing this line of investigation, levosimendan has been shown to enhance in vitro the contractility of the diaphragm muscle fibers of non-ALS patients and to improve in vivo diaphragm neuromuscular efficiency in healthy subjects. Possible positive effects on respiratory function in people with ALS were seen in an exploratory phase 2 study, and a phase 3 clinical trial is now underway to evaluate the potential benefit of an oral form of levosimendan on both respiratory and overall functions in patients with ALS. Here, we will review the various known pharmacologic effects of levosimendan, considering their relevance to people living with ALS.

Voluntary running counteracts right ventricular adverse remodeling and myocyte contraction impairment in pulmonary arterial hypertension model

AimAnalyze the effects of voluntary running during the development of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) on the right ventricle (RV) structure, RV myocyte contractility and intracellular Ca2+ transient in rats with MCT-induced PAH. Main methodsMale Wistar rats were housed sedentary or with free access to a running wheel after MCT or saline injection for until HF or median end-point day of HF in sedentary animals (24 days). Echocardiographic examination and exercise tolerance test were carried out at specific time points of the experimental period. After euthanasia, the heart was dissected, weighed and processed for either histological or single myocyte contractility and intracellular Ca2+ transient analyzes. Key findingsVoluntary running delayed the onset of HF (29 days) and the increase in pulmonary artery resistance, and improved exercise tolerance. In the median end-point day of HF, exercise retarded RV adverse remodeling (i.e. increase in extracellular matrix and collagen content). At this stage, exercise also delayed impairments in cell contractile function (i.e. amplitude and times to peak and to half relaxation) and intracellular calcium cycling (i.e. amplitude and times to peak and to half decay) in RV single myocytes. SignificanceAlong with HF onset delay and physical effort tolerance enhancement, voluntary running during the development of PAH postpones pulmonary artery resistance increases, RV adverse remodeling and myocyte contractility and intracellular calcium cycling deterioration in rats. Therefore, self-paced intermittent exercise of high intensity may contribute positively to the health and survival of individuals with PAH.

P1593VEGFR1-PLC signaling is implicated in normal spontaneous firing of cardiac pacemaker cells

Abstract Spontaneous firing of sinoatrial node cells (SANC) is regulated by sarcoplasmic reticulum (SR) generated local subsarcolemmal calcium releases (LCRs). LCRs appear during diastolic depolarization (DD) and activate an inward sodium-calcium exchange current to accelerate the DD rate and thus spontaneous SANC firing. Vascular endothelial growth factor (VEGF) receptors VEGFR1 and VEGFR2 activate PLC, and suppression of VEGFR-PLC signaling decreases calcium transients and contractility in ventricular myocytes. We tested the idea that VEGFR-PLC signaling may contribute to normal spontaneous beating of SANC. We observed that expression of VEGFR1 (assessed by RT-qPCR) in rabbit sinoatrial node was comparable to that of β1-adrenergic receptors, but less than that in ventricle. The pan VEGFR1/2/3 inhibitor PTK787/ZK222584 (10 μmol/L) in a time-dependent manner: (1) suppressed spontaneous SANC beating rate by ∼50% (perforated patch-clamp technique); (2) markedly decreased the LCR size and number per each spontaneous cycle (confocal microscopy, calcium indicator Fluo-3) and prolonged the LCR period (the interval between action potential-induced calcium transient and occurrence of subsequent LCR). The PTK787/ZK222584-induced increase in the LCR period (from 348.2±30.3 to 619.9±103.4 msec; P&lt;0.05) predicted the concomitant increase in the spontaneous cycle length (from 405.6±31.1 to 702.1±105.1 msec; P&lt;0.05), suggesting that calcium cycling could be a major target of VEGFR-dependent regulation of SANC firing. All effects of PTK787/ZK222584 were reversible upon washout. To elucidate whether signaling of VEGFR1 or VEGFR2 regulated spontaneous SANC firing, we employed a selective VEGFR2 inhibitor ZM-323881 (5 μmol/L), which suppressed spontaneous beating rate in only 2 of 10 SANC. These results indicate that VEGFR1, but not VEGFR2, is likely a key receptor that modulates automaticity in majority of SANC. To clarify downstream targets from VEGFR1 we employed PLC inhibitor U-73122, which decreased the LCR size, number and prolonged the LCR period. The inactive analog U-73343 was without effect. Because LCRs are critically dependent upon the SR calcium load, supplied by L-type calcium current (ICa,L), we examined effects of U-73122 on ICa,L. U-73122, but not U-73343, markedly suppressed ICa,L amplitude by ∼50%, leading to a decrease in the calcium influx and, as a result, to decrease in the LCR parameters, prolongation of the LCR period and spontaneous SANC cycle length. Thus, basal VEGFR1 signaling activates PLC, which modulates intracellular SR calcium cycling and LCR characteristics in SANC. We conclude that VEGFR1-PLC is a novel mechanism involved in the regulation of normal automaticity of cardiac pacemaker cells. Acknowledgement/Funding Intramural Research Program, National Institute on Aging, NIH

Ca2+-independent but voltage-dependent quantal catecholamine secretion (CiVDS) in the mammalian sympathetic nervous system

Action potential-induced vesicular exocytosis is considered exclusively Ca2+ dependent in Katz's Ca2+ hypothesis on synaptic transmission. This long-standing concept gets an exception following the discovery of Ca2+-independent but voltage-dependent secretion (CiVDS) and its molecular mechanisms in dorsal root ganglion sensory neurons. However, whether CiVDS presents only in sensory cells remains elusive. Here, by combining multiple independent recordings, we report that [1] CiVDS robustly presents in the sympathetic nervous system, including sympathetic superior cervical ganglion neurons and slice adrenal chromaffin cells, [2] uses voltage sensors of Ca2+ channels (N-type and novel L-type), and [3] contributes to catecholamine release in both homeostatic and fight-or-flight like states; [4] CiVDS-mediated catecholamine release is faster than that of Ca2+-dependent secretion at the quantal level and [5] increases Ca2+ currents and contractility of cardiac myocytes. Together, CiVDS presents in the sympathetic nervous system with potential physiological functions, including cardiac muscle contractility.