Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

An objective tool for quantifying atrial fibrillation substrate in rats.

The utility of rodents for research related to atrial fibrillation (AF) is growing exponentially. However, the obtained arrhythmic waveforms are often mixed with ventricular signals and the ability to analyze regularity and complexity of such events is limited. Recently, we introduced an implantable quadripolar electrode adapted for advanced atrial electrophysiology in ambulatory rats. Notably, we have found that the implantation itself leads to progressive atrial remodeling, presumably because of mechanical loading of the atria. In the present study, we developed an algorithm to clean the atrial signals from ventricular mixing and thereafter quantify the AF substrate in an objective manner based on waveform complexity. Rats were sequentially examined 1-, 4-, and 8-wk postelectrode implantation using a standard AF triggering protocol. Preburst ventricular mixing was sampled and automatically subtracted based on QRS detection in the ECG. Thereafter, the "pure" atrial signals were analyzed by Lempel-Ziv complexity algorithm and a complexity ratio (CR) was defined for each signal by normalizing the postburst to the preburst values. Receiver operating characteristic (ROC) curve analysis indicated an optimal CR cutoff of 1.236 that detected irregular arrhythmic events with high sensitivity (94.5%), specificity (93.1%), and area under the curve (AUC) (0.96, 95% confidence interval, 0.945-0.976). Automated and unbiased analysis indicated a gradual increase in signal complexity over time with augmentation of high frequencies in power spectrum analysis. Our findings indicate that CR algorithm detects irregularity in a highly efficient manner and can also detect the atrial remodeling induced by electrode implantation. Thus, CR analysis can strongly facilitate standardized AF research in rodents.NEW & NOTEWORTHY Rodents are increasingly used in AF research. However, because of technical difficulties including atrial waveform mixing by ventricular signals, most studies do not discriminate between irregular (i.e., AF) and regular atrial arrhythmias. Here, we develop an unbiased computerized tool to "pure" the atrial signals from ventricular mixing and thereafter analyze AF substrate based on the level of irregularity in an objective manner. This novel tool can facilitate standardized AF research in rodents.

Functional defects in hiPSCs-derived cardiomyocytes from patients with a PLEKHM2-mutation associated with dilated cardiomyopathy and left ventricular non-compaction.

Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and ß-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient's iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.

A Novel Recessive Mutation in SPEG Causes Early Onset Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common cause of heart failure and sudden cardiac death. It has been estimated that up to half of DCM cases are hereditary. Mutations in more than 50 genes, primarily autosomal dominant, have been reported. Although rare, recessive mutations are thought to contribute considerably to DCM, especially in young children. Here we identified a novel recessive mutation in the striated muscle enriched protein kinase (SPEG, p. E1680K) gene in a family with nonsyndromic, early onset DCM. To ascertain the pathogenicity of this mutation, we generated SPEG E1680K homozygous mutant human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) using CRISPR/Cas9-mediated genome editing. Functional studies in mutant iPSC-CMs showed aberrant calcium homeostasis, impaired contractility, and sarcomeric disorganization, recapitulating the hallmarks of DCM. By combining genetic analysis with human iPSCs, genome editing, and functional assays, we identified SPEG E1680K as a novel mutation associated with early onset DCM and provide evidence for its pathogenicity in vitro. Our study provides a conceptual paradigm for establishing genotype-phenotype associations in DCM with autosomal recessive inheritance.

Recapitulation of dyssynchrony-associated contractile impairment in asymmetrically paced engineered heart tissue.

BACKGROUND: One third of heart failure patients exhibit dyssynchronized electromechanical activity of the heart (evidenced by a broad QRS-complex). Cardiac resynchronization therapy (CRT) in the form of biventricular pacing improves cardiac output and clinical outcome of responding patients. Technically demanding and laborious large animal models have been developed to better predict responders of CRT and to investigate molecular mechanisms of dyssynchrony and CRT. The aim of this study was to establish a first humanized in vitro model of dyssynchrony and CRT. METHODS: Cardiomyocytes were differentiated from human induced pluripotent stem cells and cast into a fibrin matrix to produce engineered heart tissue (EHT). EHTs were either field stimulated in their entirety (symmetrically) or excited locally from one end (asymmetrically) or they were allowed to beat spontaneously. RESULTS: Asymmetrical pacing led to a depolarization wave from one end to the other end, which was visualized in human EHT transduced with a fast genetic Ca2+-sensor (GCaMP6f) arguing for dyssynchronous excitation. Symmetrical pacing in contrast led to an instantaneous (synchronized) Ca2+-signal throughout the EHT. To investigate acute and long-term functional effects, spontaneously beating human EHTs (0.5-0.8 Hz) were divided into a non-paced control group, a symmetrically and an asymmetrically paced group, each stimulated at 1 Hz. Symmetrical pacing was clearly superior to asymmetrical pacing or no pacing regarding contractile force both acutely and even more pronounced after weeks of continuous stimulation. Contractile dysfunction that can be evoked by an increased afterload was aggravated in the asymmetrically paced group. Consistent with reports from paced dogs, p38MAPK and CaMKII-abundance was higher under asymmetrical than under symmetrical pacing while pAKT was considerably lower. CONCLUSIONS: This model allows for long-term pacing experiments mimicking electrical dyssynchrony vs. synchrony in vitro. Combined with force measurement and afterload stimulus manipulation, it provides a robust new tool to gain insight into the biology of dyssynchrony and CRT.

Dock10 Regulates Cardiac Function under Neurohormonal Stress.

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.

A new implantable tool for repeated assessment of supraventricular electrophysiology and atrial fibrillation susceptibility in freely moving rats.

The complex pathophysiology of atrial fibrillation (AF) is governed by multiple risk factors in ways that are still elusive. Basic electrophysiological properties, including atrial effective refractory period (AERP) and conduction velocity, are major factors determining the susceptibility of the atrial myocardium to AF. Although there is a great need for affordable animal models in this field of research, in vivo rodent studies are limited by technical challenges. Recently, we introduced an implantable system for long-term assessment of AF susceptibility in ambulatory rats. However, technical considerations did not allow us to perform concomitant supraventricular electrophysiology measurements. Here, we designed a novel quadripolar electrode specifically adapted for comprehensive atrial studies in ambulatory rats. Electrodes were fabricated from medical-grade silicone, four platinum-iridium poles, and stainless-steel fixating pins. Initial quality validation was performed ex vivo, followed by implantation in adult rats and repeated electrophysiological studies 1, 4, and 8 wk postimplantation. Capture threshold was stable. Baseline AERP values (38.1 ± 2.3 and 39.5 ± 2.0 using 70-ms and 120-ms S1-S1 cycle lengths, respectively) confirmed the expected absence of rate adaptation in the unanesthetized state and validated our prediction that markedly higher values reported under anesthesia are nonphysiological. Evaluation of AF substrate in parallel with electrophysiological parameters validated our recent finding of a gradual increase in AF susceptibility over time and demonstrated that this phenomenon is associated with an electrical remodeling process characterized by AERP shortening. Our findings indicate that the miniature quadripolar electrode is a potent new tool, which opens a window of opportunities for better utilization of rats in AF research.NEW & NOTEWORTHY Rodents are increasingly used in AF research. However, technical challenges restrict long-term supraventricular electrophysiology studies in these species. Here, we developed an implantable electrode adapted for such studies in the rat. Our findings indicate that this new tool is effective for long-term follow-up of critical parameters such as atrial refractoriness. Obtained data shed light on the normal electrophysiology and on the increased AF susceptibility that develops in rats with implanted atrial electrodes over time.

The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells.

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell's morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.

CAP2 mutation leads to impaired actin dynamics and associates with supraventricular tachycardia and dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure and excessive risk of sudden cardiac death. Around half of DCM cases are idiopathic, and genetic factors seem to play an important role. AIM: We investigated a possible genetic cause of DCM in two consanguineous children from a Bedouin family. METHODS AND RESULTS: Using exome sequencing and searching for rare homozygous variations, we identified a nucleotide change in the donor splice consensus sequence of exon 7 in CAP2 as the causative mutation. Using patient-derived fibroblasts, we demonstrated that the mutation causes skipping of exons 6 and 7. The resulting protein is missing 64 amino acids in its N-CAP domain that should prevent its correct folding. CAP2 protein level was markedly reduced without notable compensation by the homolog CAP1. However, ß-actin mRNA was elevated as demonstrated by real-time qPCR. In agreement with the essential role of CAP2 in actin filament polymerization, we demonstrate that the mutation affects the kinetics of repolymerization of actin in patient fibroblasts. CONCLUSIONS: This is the first report of a recessive deleterious mutation in CAP2 and its association with DCM in humans. The clinical phenotype recapitulates the damaging effects on the heart observed in Cap2 knockout mice including DCM and cardiac conduction disease, but not the other effects on growth, viability, wound healing and eye development. Our data underscore the importance of the proper kinetics of actin polymerization for normal function of the human heart.

Nanoparticle Delivery of miRNA-21 Mimic to Cardiac Macrophages Improves Myocardial Remodeling after Myocardial Infarction.

MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from pro-inflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA-21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21 with a potential for use to attenuate post-MI remodeling and heart failure.

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.

Plekhm2 acts as an autophagy modulator in murine heart and cardiofibroblasts.

Plekhm2 is a protein regulating endosomal trafficking and lysosomal distribution. We recently linked a recessive inherited mutation in PLEKHM2 to a familial form of dilated cardiomyopathy and left ventricular non-compaction. These patients' primary fibroblasts exhibited abnormal lysosomal distribution and autophagy impairment. We therefore hypothesized that loss of PLEKHM2 impairs cardiac function via autophagy derangement. Here, we characterized the roles of Plekhm2 in the heart using global Plekhm2 knockout (PLK2-KO) mice and cultured cardiac cells. Compared to littermate controls (WT), young PLK2-KO mice exhibited no difference in heart function or autophagy markers but demonstrated higher basal AKT phosphorylation. Older PLK2-KO mice had body and heart growth retardation and increased LC3II protein levels. PLK2-KO mice were more vulnerable to fasting and, interestingly, impaired autophagy was noted in vitro, in Plekhm2-deficient cardiofibroblasts but not in cardiomyocytes. PLK2-KO hearts appeared to be less sensitive to pathological hypertrophy induced by angiotensin-II compared to WT. Our findings suggest a role of Plekhm2 in murine cardiac autophagy. Plekhm2 deficiency impaired autophagy in cardiofibroblasts, but the autophagy in cardiomyocytes is not critically dependent on Plekhm2. The absence of Plekhm2 in mice appears to promote compensatory mechanism(s) enabling the heart to manage angiotensin-II-induced stress without detrimental consequences.

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

INO-8875, a highly selective A1 adenosine receptor agonist: evaluation of chronotropic, dromotropic, and hemodynamic effects in rats.

Selective pharmacological activation of the adenosine 1 receptor (A(1)R) is a promising new approach to achieve a potent block of atrioventricular (A-V)-nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A(1)R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V-nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 µM) progressively prolonged A-V-nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 µg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1-50 µg/kg) and CVT-510 (20 and 50 µg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V-nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V-nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A(1)R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.

Cannabinoid receptor agonist attenuates angiotensin II-induced enlargement and mitochondrial dysfunction in rat atrial cardiomyocytes.

Pathological remodeling of atrial tissue renders the atria more prone to arrhythmia upon arrival of electrical triggers. Activation of the renin-angiotensin system is an important factor that contributes to atrial remodeling, which may result in atrial hypertrophy and prolongation of P-wave duration. In addition, atrial cardiomyocytes are electrically coupled via gap junctions, and electrical remodeling of connexins may result in dysfunction of coordinated wave propagation within the atria. Currently, there is a lack of effective therapeutic strategies that target atrial remodeling. We previously proposed that cannabinoid receptors (CBR) may have cardioprotective qualities. CB13 is a dual cannabinoid receptor agonist that activates AMPK signaling in ventricular cardiomyocytes. We reported that CB13 attenuates tachypacing-induced shortening of atrial refractoriness and inhibition of AMPK signaling in the rat atria. Here, we evaluated the effects of CB13 on neonatal atrial rat cardiomyocytes (NRAM) stimulated by angiotensin II (AngII) in terms of atrial myocyte enlargement and mitochondrial function. CB13 inhibited AngII-induced enhancement of atrial myocyte surface area in an AMPK-dependent manner. CB13 also inhibited mitochondrial membrane potential deterioration in the same context. However, AngII and CB13 did not affect mitochondrial permeability transition pore opening. We further demonstrate that CB13 increased Cx43 compared to AngII-treated neonatal rat atrial myocytes. Overall, our results support the notion that CBR activation promotes atrial AMPK activation, and prevents myocyte enlargement (an indicator that suggests pathological hypertrophy), mitochondrial depolarization and Cx43 destabilization. Therefore, peripheral CBR activation should be further tested as a novel treatment strategy in the context of atrial remodeling.

ZnT1 induces a crosstalk between T-type and L-type calcium channels through interactions with Raf-1 kinase and the calcium channel ß2 subunit.

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.

Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome.

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

Incorrectly corrected? QT interval analysis in rats and mice.

QT interval, a surrogate measure for ventricular action potential duration (APD) in the surface ECG, is widely used to identify cardiac abnormalities and drug safety. In humans, cardiac APD and QT interval are prominently affected by heart rate (HR), leading to widely accepted formulas to correct the QT interval for HR changes (QT corrected - QTc). While QTc is widely used in the clinic, the proper way to correct the QT interval in small mammals such as rats and mice is not clear. Over the years, empiric correction formulas were developed for rats and mice, which are widely used in the literature. Recent experimental findings obtained from pharmacological and direct pacing experiments in unanesthetized rodents show that the rate-adaptation properties are markedly different from those in humans and the use of existing QTc formulae can lead to major errors in data interpretation. In the present review, these experimental findings are summarized and discussed.