Cardiomyocyte Ca2 Research Articles

P6279Mitochondria targeted catalase overexpression protects against beta-adrenergic receptor mediated cardiac remodeling in mice

Abstract Background Beta-adrenergic receptor signaling is widely recognized as a fundamental component in the pathogenesis of chronic heart failure. However, the mechanisms behind beta-adrenergic receptor-mediated remodeling in cardiomyocytes and the myocardium are not fully understood. Oxidative stress has been proposed as a central pathophysiological mediator in cardiovascular disease and heart failure. The triggers and sources of oxidative stress in heart failure remain unclear. In this study we use mice with mitochondria-targeted overexpression of the antioxidant enzyme catalase (mCAT) to link beta-adrenergic receptor-mediated stress to mitochondrial reactive oxidative species (ROS) and the progression of heart failure. Hypothesis Mitochondrial ROS, induced by beta-adrenergic receptor-activation, is a mediator in the progression of the heart failure phenotype. Methods mCAT and wild type mice (n=10) were administered the non-selective beta-adrenergic agonist Isoprotenerol (Iso; 50mg/kg/day) through subcutaneous osmotic pumps for 3 weeks. Hearts were taken for biochemistry (western blotting, qPCR). Cardiomyocytes were isolated and loaded with Fluo-3 AM to study intracellular Ca2+ transients and fractional shortening using confocal line scan microscopy. All experiments were performed in accordance with the Stockholm ethical committee for animal research. Results and conclusions The WT mice displayed an increased heart/body weight ratio following chronic Iso administration. In contrast, mCAT mice displayed resistance to Iso-induced cardiac hypertrophy (p<0.05). Furthermore, chronic Iso exposure in WT mice induced increased ROS-dependent post-translational protein modifications, impaired cardiomyocyte Ca2+ handling and reduced contractility in isolated cardiomyocytes (p<0.05). Cardiomyocytes from mCAT mice did not display the deleterious effects of chronic Iso exposure on cardiomyocyte Ca2+ and contractility. Our study demonstrates that beta-adrenergic receptor stimulation-induced remodeling of the heart, which is similar to what is seen in heart failure, can be prevented by overexpressing catalase in the mitochondria. This indicates an important role of mitochondrial ROS in the link between adrenergic signaling and the development of cardiomyopathy and heart failure. Acknowledgement/Funding Hjärtlungfonden

The effects of macrophages on cardiomyocyte calcium-handling function using in vitro culture models.

Following myocardial infarction (MI), myocardial inflammation plays a crucial role in the pathogenesis of MI injury and macrophages are among the key cells activated during the initial phases of the host response regulating the healing process. While macrophages have emerged as attractive effectors in tissue injury and repair, the contribution of macrophages on cardiac cell function and survival is not fully understood due to complexity of the in vivo inflammatory microenvironment. Understanding the key cells involved and how they communicate with one another is of paramount importance for the development of effective clinical treatments. Here, novel in vitro myocardial inflammation models were developed to examine how both direct and indirect interactions with polarized macrophage subsets present in the post‐MI microenvironment affect cardiomyocyte function. The indirect model using conditioned medium from polarized macrophage subsets allowed examination of the effects of macrophage‐derived factors on stem cell‐derived cardiomyocyte function for up to 3 days. The results from the indirect model demonstrated that pro‐inflammatory macrophage‐derived factors led to a significant downregulation of cardiac troponin T (cTnT) and sarcoplasmic/endoplasmic reticulum calcium ATPase (Serca2) gene expression. It also demonstrated that inhibition of macrophage‐secreted matricellular protein, osteopontin (OPN), led to a significant decrease in cardiomyocyte store‐operated calcium entry (SOCE). In the direct model, stem cell‐derived cardiomyocytes were co‐cultured with polarized macrophage subsets for up to 3 days. It was demonstrated that anti‐inflammatory macrophages significantly increased cardiomyocyte Ca2+ fractional release while macrophages independent of their subtypes led to significant downregulation of SOCE response in cardiomyocytes. This study describes simplified and controlled in vitro myocardial inflammation models, which allow examination of potential beneficial and deleterious effects of macrophages on cardiomyocytes and vise versa. This can lead to our improved understanding of the inflammatory microenvironment post‐MI, otherwise difficult to directly investigate in vivo or by using currently available in vitro models.

Impaired Activity of Ryanodine Receptors Contributes to Calcium Mishandling in Cardiomyocytes of Metabolic Syndrome Rats.

Metabolic syndrome (MetS) has become a global epidemic. MetS is a serious health problem because of its related cardiovascular complications, which include hypertension and delayed heart rate recovery after exercise. The molecular bases of cardiac dysfunction in MetS are still under scrutiny and may be related to anomalies in the activity and expression of key proteins involved in the cardiac excitation–contraction coupling (ECC). The cardiac Ca2+ channel/ryanodine receptor (RyR2) participates in releasing Ca2+ from internal stores and plays a key role in the modulation of ECC. We examined alterations in expression, phosphorylation status, Ca2+ sensitivity, and in situ function (by measuring Ca2+ sparks and Ca2+ transients) of RyR2; alterations in these characteristics could help to explain the Ca2+ handling disturbances in MetS cardiomyocytes. MetS was induced in rats by adding commercially refined sugar (30% sucrose) to their drinking water for 24 weeks. Cardiomyocytes of MetS rats displayed decreased Ca2+ transient amplitude and cell contractility at all stimulation frequencies. Quiescent MetS cardiomyocytes showed a decrease in Ca2+ spark frequency, amplitude, and spark-mediated Ca2+ leak. The [3H]-ryanodine binding data showed that functionally active RyRs are significantly diminished in MetS heart microsomes; and exhibited rapid Ca2+-induced inactivation. The phosphorylation of corresponding Ser2814 (a preferential target for CaMKII) of the hRyR2 was significantly diminished. RyR2 protein expression and Ser2808 phosphorylation level were both unchanged. Further, we demonstrated that cardiomyocyte Ca2+ mishandling was associated with reduced SERCA pump activity due to decreased Thr17-PLN phosphorylation, suggesting a downregulation of CaMKII in MetS hearts, though the SR Ca2+ load remained unchanged. The reduction in the phosphorylation level of RyR2 at Ser2814 decreases RyR2 availability for activation during ECC. In conclusion, the impaired in situ activity of RyR2 may also account for the poor overall cardiac outcome reported in MetS patients; hence, the SERCA pump and RyR2 are both attractive potential targets for future therapies.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Postnatal undernutrition alters adult female mouse cardiac structure and function leading to limited exercise capacity.

Impaired growth during fetal life can reprogramme heart development and increase the risk for long-term cardiovascular dysfunction. It is uncertain if the developmental window during which the heart is vulnerable to reprogramming as a result of inadequate nutrition extends into the postnatal period. We found that adult female mice that had been undernourished only from birth to 3weeks of age had disproportionately smaller hearts compared to males, with thinner ventricle walls and more mononucleated cardiomyocytes. In females, but not males, cardiac diastolic function, and heart rate responsiveness to adrenergic stimulation were limited and maximal exercise capacity was compromised. These data suggest that the developmental window during which the heart is vulnerable to reprogramming by inadequacies in nutrient intake may extend into postnatal life and such individuals could be at increased risk for a cardiac event as a result of strenuous exercise. Adults who experienced undernutrition during critical windows of development are at increased risk for cardiovascular disease. The contribution of cardiac function to this increased disease risk is uncertain. We evaluated the effect of a short episode of postnatal undernutrition on cardiovascular function in mice at the whole animal, organ, and cellular levels. Pups born to control mouse dams were suckled from birth to postnatal day (PN) 21 on dams fed either a control (20% protein) or a low protein (8% protein) isocaloric diet. After PN21 offspring were fed the same control diet until adulthood. At PN70 was measured by treadmill test. At PN80 cardiac function was evaluated by echocardiography and Doppler analysis at rest and following β-adrenergic stimulation. Isolated cardiomyocyte nucleation and Ca2+ transients (with and without β-adrenergic stimulation) were measured at PN90. Female mice that were undernourished and then refed (PUN), unlike male mice, had disproportionately smaller hearts and their exercise capacity, cardiac diastolic function, and heart rate responsiveness to adrenergic stimulation were limited. A reduced left ventricular end diastolic volume, impaired early filling, and decreased stored energy at the beginning of diastole contributed to these impairments. Female PUN mice had more mononucleated cardiomyocytes; under resting conditions binucleated cells had a functional profile suggestive of increased basal adrenergic activation. Thus, a brief episode of early postnatal undernutrition in the mouse can produce persistent changes to cardiac structure and function that limit exercise/functional capacity and thereby increase the risk for the development of a wide variety of cardiovascular morbidities.

Circulating Secretoneurin Concentrations After Cardiac Surgery: Data From the FINNish Acute Kidney Injury Heart Study.

Secretoneurin is associated with cardiomyocyte Ca handling and improves risk prediction in patients with acute myocardial dysfunction. Whether secretoneurin improves risk assessment on top of established cardiac biomarkers and European System for Cardiac Operative Risk Evaluation II in patients undergoing cardiac surgery is not known. Prospective, observational, single-center sub-study of a multicenter study. Prospective observational study of survival in patients undergoing cardiac surgery. A total of 619 patients undergoing cardiac surgery. Patients underwent either isolated coronary artery bypass graft surgery, single noncoronary artery bypass graft surgery, two procedures, or three or more procedures. Procedures other than coronary artery bypass graft were valve surgery, surgery on thoracic aorta, and other cardiac surgery. We measured preoperative and postoperative secretoneurin concentrations and adjusted for European System for Cardiac Operative Risk Evaluation II, N-terminal pro-B-type natriuretic peptide, and cardiac troponin T concentrations in multivariate analyses. During 961 days of follow-up, 59 patients died (9.5%). Secretoneurin concentrations were higher among nonsurvivors compared with survivors, both before (168 pmol/L [quartile 1-3, 147-206 pmol/L] vs 160 pmol/L [131-193 pmol/L]; p = 0.039) and after cardiac surgery (173 pmol/L [129-217 pmol/L] vs 143 pmol/L [111-173 pmol/L]; p < 0.001). Secretoneurin concentrations decreased from preoperative to postoperative measurements in survivors, whereas we observed no significant decrease in secretoneurin concentrations among nonsurvivors. Secretoneurin concentrations were weakly correlated with established risk indices. Patients with the highest postoperative secretoneurin concentrations had worse outcome compared with patients with lower secretoneurin concentrations (p < 0.001 by the log-rank test) and postoperative secretoneurin concentrations were associated with time to death in multivariate Cox regression analysis: hazard ratio lnsecretoneurin 2.96 (95% CI, 1.46-5.99; p = 0.003). Adding postoperative secretoneurin concentrations to European System for Cardiac Operative Risk Evaluation II improved patient risk stratification, as assessed by the integrated discrimination index: 0.023 (95% CI, 0.0043-0.041; p = 0.016). Circulating postoperative secretoneurin concentrations provide incremental prognostic information to established risk indices in patients undergoing cardiac surgery.

Phenolic Metabolites Modulate Cardiomyocyte Beating in Response to Isoproterenol

Cardiovascular disease (CVD) is a public health concern, and the third cause of death worldwide. Several epidemiological studies and experimental approaches have demonstrated that consumption of polyphenol-enriched fruits and vegetables can promote cardioprotection. Thus, diet plays a key role in CVD development and/or prevention. Physiological β-adrenergic stimulation promotes beneficial inotropic effects by increasing heart rate, contractility and relaxation speed of cardiomyocytes. Nevertheless, chronic activation of β-adrenergic receptors can cause arrhythmias, oxidative stress and cell death. Herein the cardioprotective effect of human metabolites derived from polyphenols present in berries was assessed in cardiomyocytes, in response to chronic β-adrenergic stimulation, to disclose some of the underlying molecular mechanisms. Ventricular cardiomyocytes derived from neonate rats were treated with three human bioavailable phenolic metabolites found in circulating human plasma, following berries' ingestion (catechol-O-sulphate, pyrogallol-O-sulphate, and 1-methylpyrogallol-O-sulphate). The experimental conditions mimic the physiological concentrations and circulating time of these metabolites in the human plasma (2h). Cardiomyocytes were then challenged with the β-adrenergic agonist isoproterenol (ISO) for 24h. The presence of phenolic metabolites limited ISO-induced mitochondrial oxidative stress. Likewise, phenolic metabolites increased cell beating rate and synchronized cardiomyocyte beating population, following prolonged β-adrenergic receptor activation. Finally, phenolic metabolites also prevented ISO-increased activation of PKA-cAMP pathway, modulating Ca2+ signalling and rescuing cells from an arrhythmogenic Ca2+ transients' phenotype. Unexpected cardioprotective properties of the recently identified human-circulating berry-derived polyphenol metabolites were identified. These metabolites modulate cardiomyocyte beating and Ca2+ transients following β-adrenergic prolonged stimulation.

The Dipeptidyl Peptidase 4 Substrate CXCL12 Has Opposing Cardiac Effects in Young Mice and Aged Diabetic Mice Mediated by Ca2+ Flux and Phosphoinositide 3-Kinase γ.

Blood glucose-lowering therapies can positively or negatively affect heart function in type 2 diabetes, or they can have neutral effects. Dipeptidyl peptidase 4 (DPP-4) inhibitors lower blood glucose by preventing the proteolytic inactivation of glucagon-like peptide 1 (GLP-1). However, GLP-1 is not the only peptide substrate of DPP-4. Here, we investigated the GLP-1-independent cardiac effects of DPP-4 substrates. Pointing to GLP-1 receptor (GLP-1R)-independent actions, DPP-4 inhibition prevented systolic dysfunction equally in pressure-overloaded wild-type and GLP-1R knockout mice. Likewise, DPP-4 inhibition or the DPP-4 substrates substance P or C-X-C motif chemokine ligand 12 (CXCL12) improved contractile recovery after no-flow ischemia in the hearts of otherwise healthy young adult mice. Either DPP-4 inhibition or CXCL12 increased phosphorylation of the Ca2+ regulatory protein phospholamban (PLN), and CXCL12 directly enhanced cardiomyocyte Ca2+ flux. In contrast, hearts of aged obese diabetic mice (which may better mimic the comorbid patient population) had diminished levels of PLN phosphorylation. In this setting, CXCL12 paradoxically impaired cardiac contractility in a phosphoinositide 3-kinase γ-dependent manner. These findings indicate that the cardiac effects of DPP-4 inhibition primarily occur through GLP-1R-independent processes and that ostensibly beneficial DPP-4 substrates can paradoxically worsen heart function in the presence of comorbid diabetes.

Reduced myocyte complex N-glycosylation causes dilated cardiomyopathy.

Protein glycosylation is an essential posttranslational modification that affects a myriad of physiologic processes. Humans with genetic defects in glycosylation, which result in truncated glycans, often present with significant cardiac deficits. Acquired heart diseases and their associated risk factors were also linked to aberrant glycosylation, highlighting its importance in human cardiac disease. In both cases, the link between causation and corollary remains enigmatic. The glycosyltransferase gene, mannosyl (α-1,3-)-glycoprotein β-1,2- N-acetylglucosaminyltransferase (Mgat1), whose product, N-acetylglucosaminyltransferase 1 (GlcNAcT1) is necessary for the formation of hybrid and complex N-glycan structures in the medial Golgi, was shown to be at reduced levels in human end-stage cardiomyopathy, thus making Mgat1 an attractive target for investigating the role of hybrid/complex N-glycosylation in cardiac pathogenesis. Here, we created a cardiomyocyte-specific Mgat1 knockout (KO) mouse to establish a model useful in exploring the relationship between hybrid/complex N-glycosylation and cardiac function and disease. Biochemical and glycomic analyses showed that Mgat1KO cardiomyocytes produce predominately truncated N-glycan structures. All Mgat1KO mice died significantly younger than control mice and demonstrated chamber dilation and systolic dysfunction resembling human dilated cardiomyopathy (DCM). Data also indicate that a cardiomyocyte L-type voltage-gated Ca2+ channel (Cav) subunit (α2δ1) is a GlcNAcT1 target, and Mgat1KO Cav activity is shifted to more-depolarized membrane potentials. Consistently, Mgat1KO cardiomyocyte Ca2+ handling is altered and contraction is dyssynchronous compared with controls. The data demonstrate that reduced hybrid/complex N-glycosylation contributes to aberrant cardiac function at whole-heart and myocyte levels drawing a direct link between altered glycosylation and heart disease. Thus, the Mgat1KO provides a model for investigating the relationship between systemic reductions in glycosylation and cardiac disease, showing that clinically relevant changes in cardiomyocyte hybrid/complex N-glycosylation are sufficient to cause DCM and early death.-Ednie, A. R., Deng, W., Yip, K.-P., Bennett, E. S. Reduced myocyte complex N-glycosylation causes dilated cardiomyopathy.

The Subcellular Distribution of Ryanodine Receptors and L-Type Ca2+ Channels Modulates Ca2+-Transient Properties and Spontaneous Ca2+-Release Events in Atrial Cardiomyocytes.

Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

Cardiomyocyte-specific disruption of Cathepsin K protects against doxorubicin-induced cardiotoxicity

The lysosomal cysteine protease Cathepsin K is elevated in humans and animal models of heart failure. Our recent studies show that whole-body deletion of Cathepsin K protects mice against cardiac dysfunction. Whether this is attributable to a direct effect on cardiomyocytes or is a consequence of the global metabolic alterations associated with Cathepsin K deletion is unknown. To determine the role of Cathepsin K in cardiomyocytes, we developed a cardiomyocyte-specific Cathepsin K-deficient mouse model and tested the hypothesis that ablation of Cathepsin K in cardiomyocytes would ameliorate the cardiotoxic side-effects of the anticancer drug doxorubicin. We used an α-myosin heavy chain promoter to drive expression of Cre, which resulted in over 80% reduction in protein and mRNA levels of cardiac Cathepsin K at baseline. Four-month-old control (Myh-Cre-; Ctskfl/fl) and Cathepsin K knockout (Myh-Cre+; Ctskfl/fl) mice received intraperitoneal injections of doxorubicin or vehicle, 1 week following which, body and tissue weight, echocardiographic properties, cardiomyocyte contractile function and Ca2+-handling were evaluated. Control mice treated with doxorubicin exhibited a marked increase in cardiac Cathepsin K, which was associated with an impairment in cardiac structure and function, evidenced as an increase in end-systolic and end-diastolic diameters, decreased fractional shortening and wall thickness, disruption in cardiac sarcomere and microfilaments and impaired intracellular Ca2+ homeostasis. In contrast, the aforementioned cardiotoxic effects of doxorubicin were attenuated or reversed in mice lacking cardiac Cathepsin K. Mechanistically, Cathepsin K-deficiency reconciled the disturbance in cardiac energy homeostasis and attenuated NF-κB signaling and apoptosis to ameliorate doxorubicin-induced cardiotoxicity. Cathepsin K may represent a viable drug target to treat cardiac disease.

Catestatin: A Master Regulator of Cardiovascular Functions

Cardiovascular disease (CVD), the most common cause of death globally, accounts for ~30% of all deaths worldwide. Hypertension is a common contributor to morbidity and mortality from CVD. The plasma concentration of chromogranin A (CgA) is elevated in patients with CVD as well as patients with established human essential hypertension and heart failure (HF). In contrast, the plasma level of the CgA-derived peptide catestatin (CST) is diminished in human essential hypertension. Low conversion of CgA-to-CST has been associated with increased mortality in patients hospitalized with acute HF. Consistent with human findings, the lack of CST in CgA knockout (Chga-KO) mice eventuates in the development of hypertension and supplementation of CST to Chga-KO mice restores blood pressure, implicating CST as a key player in regulating hypertension. In the peripheral system, CST decreases blood pressure by stimulating histamine release, inhibiting catecholamine secretion, or causing vasodilation. Centrally, CST improves baroreflex sensitivity (BRS) and heart rate variability (HRV) by exciting GABAergic neurons in the caudal ventrolateral medulla (CVLM) and pyramidal neurons of the central amygdala; CST also decreases BRS by exciting glutamatergic rostral ventrolateral medulla (RVLM) neurons. In addition, CST provides cardioprotection by inhibiting inotropy and lusitropy; activating mitochondrial KATP channels, and stimulating reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways and consequent inhibition of mitochondrial permeability transition pore (mPTP). CST modulates cardiomyocyte Ca2+ levels by direct inhibition of Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) activity and consequent reduction in phosphorylation of phospholamban and ryanodine receptor 2, thereby providing support for a direct functional role of CST in the failing myocardium. These multitude of effects establish CST as a master regulator of cardiovascular functions.

Correcting Calcium Dysregulation in Chronic Heart Failure Using SERCA2a Gene Therapy.

Chronic heart failure (CHF) is a major contributor to cardiovascular disease and is the leading cause of hospitalization for those over the age of 65, which is estimated to account for close to seventy billion dollars in healthcare costs by 2030 in the US alone. The successful therapies for preventing and reversing CHF progression are urgently required. One strategy under active investigation is to restore dysregulated myocardial calcium (Ca2+), a hallmark of CHF. It is well established that intracellular Ca2+ concentrations are tightly regulated to control efficient myocardial systolic contraction and diastolic relaxation. Among the many cell surface proteins and intracellular organelles that act as the warp and woof of the regulatory network controlling intracellular Ca2+ signals in cardiomyocytes, sarco/endoplasmic reticulum Ca2+ ATPase type 2a (SERCA2a) undoubtedly plays a central role. SERCA2a is responsible for sequestrating cytosolic Ca2+ back into the sarcoplasmic reticulum during diastole, allowing for efficient uncoupling of actin-myosin and subsequent ventricular relaxation. Accumulating evidence has demonstrated that the expression of SERCA2a is downregulated in CHF, which subsequently contributes to severe systolic and diastolic dysfunction. Therefore, restoring SERCA2a expression and improving cardiomyocyte Ca2+ handling provides an excellent alternative to currently used transplantation and mechanical assist devices in the treatment of CHF. Indeed, advancements in safe and effective gene delivery techniques have led to the emergence of SERCA2a gene therapy as a potential therapeutic choice for CHF patients. This mini-review will succinctly detail the progression of SERCA2a gene therapy from its inception in plasmid and animal models, to its clinical trials in CHF patients, highlighting potential avenues for future work along the way.

Measuring Cardiomyocyte Contractility and Calcium Handling In Vitro.

In vitro measurements of cardiomyocyte contractility and Ca2+ handling have been used as a platform for determining physiological consequence of various genetic manipulations and identifying potential therapeutic targets for the treatment of heart failure. The Myocyte Calcium and Contractility System (IonOptix) offers a simultaneous trace of sarcomere movements and changes of intracellular Ca2+ levels in a single cardiomyocyte. Herein, we describe a modified protocol for the isolation of adult cardiomyocytes from murine hearts and provide a step-by-step description on how to analyze cardiomyocyte Ca2+ transient and contractility data collected using the IonOptix system. In our modified protocol, we recommend a novel cannulation technique which simplifies this difficult method and leads to improved viability of isolated cardiomyocytes. In addition, a comprehensive analysis of intracellular Ca2+ handling, SR Ca2+ load, myofilament Ca2+ sensitivity, and cardiomyocyte contractility is described in order to provide important insights into myocardial mechanics.

Improvement of cardiomyocyte function by a novel pyrimidine-based CaMKII-inhibitor

ObjectivePathologically increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the associated Ca2+-leak from the sarcoplasmic reticulum are recognized to be important novel pharmacotherapeutic targets in heart failure and cardiac arrhythmias. However, CaMKII-inhibitory compounds for therapeutic use are still lacking. We now report on the cellular and molecular effects of a novel pyrimidine-based CaMKII inhibitor developed towards clinical use. Methods and resultsOur findings demonstrate that AS105 is a high-affinity ATP-competitive CaMKII-inhibitor that by its mode of action is also effective against autophosphorylated CaMKII (in contrast to the commonly used allosteric CaMKII-inhibitor KN-93). In isolated atrial cardiomyocytes from human donors and ventricular myocytes from CaMKIIδC-overexpressing mice with heart failure, AS105 effectively reduced diastolic SR Ca2+ leak by 38% to 65% as measured by Ca2+-sparks or tetracaine-sensitive shift in [Ca2+]i. Consistent with this, we found that AS105 suppressed arrhythmogenic spontaneous cardiomyocyte Ca2+-release (by 53%). Also, the ability of the SR to accumulate Ca2+ was enhanced by AS105, as indicated by improved post-rest potentiation of Ca2+-transient amplitudes and increased SR Ca2+-content in the murine cells. Accordingly, these cells had improved systolic Ca2+-transient amplitudes and contractility during basal stimulation. Importantly, CaMKII inhibition did not compromise systolic fractional Ca2+-release, diastolic SR Ca2+-reuptake via SERCA2a or Ca2+-extrusion via NCX. ConclusionAS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca2+-leak, thus improving SR Ca2+-accumulation and reducing cellular arrhythmogenic correlates, without negatively influencing excitation-contraction coupling. These findings further validate CaMKII as a key target in cardiovascular disease, implicated by genetic, allosteric inhibitors, and pseudo-substrate inhibitors.

Suppression of Arrhythmia by Enhancing Mitochondrial Ca2+ Uptake in Catecholaminergic Ventricular Tachycardia Models

Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca2+ handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca2+ handling. Therefore, intracellular Ca2+ transporters are lead candidate structures for novel and safer antiarrhythmic therapies. Mitochondria and mitochondrial Ca2+ transport proteins are important regulators of cardiac Ca2+ handling. Here we evaluated the potential of pharmacological activation of mitochondrial Ca2+ uptake for the treatment of cardiac arrhythmia. To this aim,we tested substances that enhance mitochondrial Ca2+ uptake for their ability to suppress arrhythmia in a murine model for ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT) in vitro and in vivo and in induced pluripotent stem cell-derived cardiomyocytes from a CPVT patient. In freshly isolated cardiomyocytes of RyR2R4496C/WT mice efsevin, a synthetic agonist of the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane, prevented the formation of diastolic Ca2+ waves and spontaneous action potentials. The antiarrhythmic effect of efsevin was abolished by blockade of the mitochondrial Ca2+ uniporter (MCU), but could be reproduced using the natural MCU activator kaempferol. Both mitochondrial Ca2+ uptake enhancers (MiCUps), efsevin and kaempferol, significantly reduced episodes of stress-induced ventricular tachycardia in RyR2R4496C/WT mice in vivo and abolished diastolic, arrhythmogenic Ca2+ events in human iPSC-derived cardiomyocytes.

Glycosylated Chromogranin A: Potential Role in the Pathogenesis of Heart Failure.

Endocrine and paracrine factors influence the cardiovascular system and the heart by a number of different mechanisms. The chromogranin-secretogranin (granin) proteins seem to represent a new family of proteins that exerts both direct and indirect effects on cardiac and vascular functions. The granin proteins are produced in multiple tissues, including cardiac cells, and circulating granin protein concentrations provide incremental prognostic information to established risk indices in patients with myocardial dysfunction. In this review, we provide recent data for the granin proteins in relation with cardiovascular disease, and with a special focus on chromogranin A and heart failure. Chromogranin A is the most studied member of the granin protein family, and shorter, functionally active peptide fragments of chromogranin A exert protective effects on myocardial cell death, ischemia-reperfusion injury, and cardiomyocyte Ca2+ handling. Granin peptides have also been found to induce angiogenesis and vasculogenesis. Protein glycosylation is an important post-translational regulatory mechanism, and we recently found chromogranin A molecules to be hyperglycosylated in the failing myocardium. Chromogranin A hyperglycosylation impaired processing of full-length chromogranin A molecules into physiologically active chromogranin A peptides, and patients with acute heart failure and low rate of chromogranin A processing had increased mortality compared to other acute heart failure patients. Other studies have also demonstrated that circulating granin protein concentrations increase in parallel with heart failure disease stage. The granin protein family seems to influence heart failure pathophysiology, and chromogranin A hyperglycosylation could directly be implicated in heart failure disease progression.

Exercise Training Protects Cardiomyocytes from Deleterious Effects of Palmitate.

We investigated the effects of palmitate, a high saturated fat, on Ca2+, action potential and reactive oxygen species dynamics in cardiomyocytes from untrained and trained mice. Male mice were subjected to moderate intensity exercise training on a treadmill. Cardiomyocytes of untrained and trained mice were isolated, treated for 30 min with palmitate and intracellular calcium transient and action potential duration were recorded. Additionally, we assessed reactive oxygen species generation. Treatment of cardiomyocytes from untrained mice with palmitate induced a significant decrease in Ca2+ transient magnitude by 34%. Exercise training did not change cardiomyocyte Ca2+ dynamics in the control group. However, trained cardiomyocytes were protected from deleterious effects of palmitate. Action potential duration was not altered by palmitate in either untrained or trained cardiomyocytes. Moreover, palmitate treatment increased reactive oxygen species generation in both untrained and trained cardiomyocytes. Nevertheless, the levels of reactive oxygen species in trained cardiomyocytes treated with palmitate were still 27% lower than those seen at basal conditions in untrained cardiomyocytes. Taken together, these findings indicate that exercise training protects cardiomyocytes from deleterious effects of palmitate possibly by inhibiting exacerbated ROS production.

Inhibition of mevalonate pathway prevents ischemia-induced cardiac dysfunction in rats via RhoA-independent signaling pathway.

We previously demonstrated that anoxia-mediated Ca2+ handling dysfunction could be ameliorated through inhibition of mevalonate pathway via RhoA- and Ras-related mechanisms in H9c2 cells. In this study, we further explored whether inhibition of mevalonate pathway is associated with cardiac remodeling and dysfunction in ischemic cardiomyopathy, and discussed the possible role of Ras, Rac and RhoA in cardiac dysfunction. We investigated the role of mevalonate pathway in cardiac remodeling and cardiomyocyte Ca2+ handling proteins expression in a rat model of cardiac dysfunction due to myocardial infarction (MI). After MI, adult male Sprague-Dawley rats were treated with drugs that antagonize key components in mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, and Rho-kinase for 10weeks. The protein expression of ryanodine receptor 2 (RyR2), sarcoplasmic reticulum Ca2+ ATPase (SERCA) 2a, phospholamban (PLB), phospho-PLB at serine-16 (PSer16-PLB), FKBP12.6, and RhoA as well as RyR2 and FKBP12.6 mRNA levels was evaluated. Rosuvastatin and alendronate treatment prevented myocardial remodeling, improved cardiac function and reduced infarct size. Furthermore, rosuvastatin and alendronate promoted an increase in the protein expression of SERCA2a and PSer16-PLB/PLB ratio as well as partially restored the RyR2 and FKBP12.6 gene and protein expression. Fasudil failed to exert these beneficial effects. These findings indicate that mevalonate pathway inhibition by rosuvastatin and alendronate prevents cardiac remodeling and dysfunction possibly through RhoA-independent mechanisms.

Chronic in vivo nitric oxide deficiency impairs cardiac functional recovery after ischemia in female (but not male) mice

Nitric oxide (NO) is an important regulator of cardiac function and plays a key role in ischemic cardioprotection. The role of chronic NO deficiency in coordinating ischemic vulnerability in female myocardium has not been established. The aim of this study was to determine the influence of chronic in vivo NO synthase inhibition in modulating ex vivo ischemia-reperfusion responses in female hearts (relative to males). Mice were subjected to l-NAME (l-NG-Nitroarginine-methyl-ester) treatment in vivo for 8weeks. Cardiac fibrotic, inflammatory and cardiomyocyte Ca2+ handling related gene expression changes were assessed. Hearts were Langendorff-perfused, subjected to 20min global ischemia with 45min reperfusion. In response to this moderate ex vivo ischemic insult, hearts derived from l-NAME treated female animals exhibited increased incidence of reperfusion arrhythmias, diastolic abnormality and reduced contractile recovery in reperfusion. This differential response was observed even though baseline performance of hearts from l-NAME treated animals was not different to vehicle controls, myocardial inflammatory and fibrotic indices were similar in males and females and the systolic blood pressure effect of l-NAME administration was equivalent in both sexes. Evaluation of a subgroup of mice with cardiomyocyte specific mineralocorticoid receptor deletion suggests involvement of this receptor in NO-deficiency mediated responses. To examine underlying pre-disposing mechanisms, expression of a panel of candidate genes encoding proteins involved in electromechanical homeostasis (particularly relevant to ischemic challenge) was evaluated in normoxic myocardial tissues from the l-NAME- and vehicle-treated animals. Analysis revealed that l-NAME treatment in females selectively regulated expression of genes related directly and indirectly to cardiomyocyte Ca2+ handling in a manner consistent with destabilization of Ca2+ homeostasis and arrhythmogenesis. Our investigation provides new insight into the role of sustained decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge.