Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor

β(1)- and β(2)-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β(2)-adrenergic receptors (β(2)ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β(2)ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β(2)AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β(2)AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation.     

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases: Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases

The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed.     

Cholesteryl ester transfer protein inhibition as a strategy to reduce cardiovascular risk

Human and rabbit plasma contain a cholesteryl ester transfer protein (CETP) that promotes net mass transfers of cholesteryl esters from high density lipoproteins (HDL) to other plasma lipoprotein fractions. As predicted, inhibition of CETP in both humans and rabbits increases the concentration of cholesterol in the potentially protective HDL fraction, while decreasing it in potentially proatherogenic non-HDL fractions. Inhibition of CETP in rabbits also inhibits the development of diet-induced atherosclerosis. However, use of the CETP inhibitor torcetrapib in humans did not reduce atheroma in three imaging trials and caused an excess of deaths and cardiovascular events in a large clinical outcome trial. The precise explanation for the harm caused by torcetrapib is unknown but may relate to documented, potentially harmful effects unrelated to inhibition of CETP. More recently, a trial using the weak CETP inhibitor dalcetrapib, which raises HDL levels less effectively than torcetrapib and does not lower non-HDL lipoprotein levels, was terminated early for reasons of futility. There was no evidence that dalcetrapib caused harm in that trial. Despite these setbacks, the hypothesis that CETP inhibitors will be antiatherogenic in humans is still being tested in studies with anacetrapib and evacetrapib, two CETP inhibitors that are much more potent than dalcetrapib and that do not share the off-target adverse effects of torcetrapib.     

Impaired redox signaling and mitochondrial uncoupling contributes vascular inflammation and cardiac dysfunction in type 1 diabetes: Protective role of arjunolic acid

Vascular inflammation and cardiac dysfunction are the leading causes of mortality and morbidity among the diabetic patients. Type 1 diabetic mellitus (T1DM) is associated with increased cardiovascular complications at an early stage of the disease. The purpose of the present study was to explore whether arjunolic acid (AA) plays any protective role against cardiovascular complications in T1DM and if so, what molecular pathways it utilizes for the mechanism of its protective action. Streptozotocin (STZ) was used to induce T1DM in experimental rats. Alteration in plasma lipid profile and release of membrane bound enzymes like LDH (lactate dehydrogenase) and CK (creatine kinase) established the association of hyperlipidemia and cell membrane disintegration with hyperglycemia. Hyperglycemia altered the levels of oxidative stress related biomarkers, decreased the intracellular NAD and ATP concentrations. Hyperglycemia-induced enhanced levels of VEGF, ICAM-1, MCP-1 and IL-6 in the plasma of STZ treated animals indicate vascular inflammation in T1DM. Histological studies and FACS analysis revealed that hyperglycemia caused cell death mostly via the apoptotic pathway. Investigating molecular mechanism, we observed NF-κB and MAPKs (p38 and ERK1/2) activations, mitochondrial membrane depolarization, cytochrome C release, caspase 3 activation and PARP cleavage in apoptotic cell death in the diabetic cardiac tissue. Treatment with AA (20 mg/kg body weight) reduced hyperglycemia, membrane disintegration, oxidative stress, vascular inflammation and prevented the activation of oxidative stress induced signaling cascades leading to cell death. Results suggest that AA possesses the potential to be a beneficial therapeutic agent in diabetes and its associated cardiac complications.     

Metabolic effects of anti-angiogenic therapy in tumors

Anti-angiogenic therapy has recently been added to the panel of cancer therapeutics, but predictive biomarkers of response are still not available. In animal models, anti-angiogenic therapy causes tumor starvation by increasing hypoxia and impairing nutrients supply. It is thus conceivable that angiogenesis inhibition causes remarkable metabolic perturbations in tumors, although they remain largely uncharted. We review here recent acquisitions about metabolic effects of angiogenesis blockade in tumors and discuss the possibility that some metabolic features of tumor cells - i.e. their dependency from glucose as primary energy substrate - might affect tumor responses to anti-VEGF treatment.     

Chromatin-modifying agents in anti-cancer therapy

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.     

A natural compound induced cardiogenic differentiation of endogenous MSCs for repair of infarcted heart

An intra-myocardial injection of a cardiogenic factor (cardiogenin) was reported to induce myocardial regeneration of exogenous mesenchymal stem cell (MSCs) origin. In this study, replacement of the dangerous intra-myocardial injection with a safe method and whether the endogenous MSCs contribute to the cardiogenin-mediated myocardial regeneration were investigated. Bone marrow transplantation with labeled MSCs was performed in rats, which were subsequently subject to a permanent ligation of left anterior descending coronary artery one week after the transplantation. The rats were then treated with the cardiogenin through oral administration for 2 weeks. We not only demonstrated the substantial therapeutic effects of cardiogenin on myocardial infarction through an oral administration, but also provided direct evidences that the bone marrow derived endogenous MSCs are the major cellular source of the regenerating myocardium. Preliminary mechanistic studies suggested that miR-9 and its target E-cadherin may be required for intercalated disc formation.