Murine Ventricular Cardiomyocytes Research Articles

Reduction of SR Ca2+ leak and arrhythmogenic cellular correlates by SMP-114, a novel CaMKII inhibitor with oral bioavailability.

Sarcoplasmic reticulum (SR) Ca2+ leak induced by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is centrally involved in atrial and ventricular arrhythmogenesis as well as heart failure remodeling. Consequently, treating SR Ca2+ leak has been proposed as a novel therapeutic paradigm, but compounds for use in humans are lacking. SMP-114 ("Rimacalib") is a novel, orally available CaMKII inhibitor developed for human use that has already entered clinical phase II trials to treat rheumatoid arthritis. We speculated that SMP-114 might also be useful to treat cardiac SR Ca2+ leak. SMP-114 significantly reduces SR Ca2+ leak (as assessed by Ca2+ sparks) in human atrial (0.72±0.33 sparks/100µm/s vs. control 3.02±0.91 sparks/100µm/s) and failing left ventricular (0.78±0.23 vs. 1.69±0.27 sparks/100µm/s) as well as in murine ventricular cardiomyocytes (0.30±0.07 vs. 1.50±0.28sparks/100µm/s). Associated with lower SR Ca2+ leak, we found that SMP-114 suppressed the occurrence of spontaneous arrhythmogenic spontaneous Ca2+ release (0.356±0.109 vs. 0.927±0.216 events per 30s stimulation cessation). In consequence, post-rest potentiation of Ca2+-transient amplitude (measured using Fura-2) during the 30s pause was improved by SMP-114 (52±5 vs. 37±4%). Noteworthy, SMP-114 has these beneficial effects without negatively impairing global excitation-contraction coupling: neither systolic Ca2+ release nor single cell contractility was compromised, and also SR Ca2+ reuptake, in line with resulting cardiomyocyte relaxation, was not impaired by SMP-114 in our assays. SMP-114 demonstrated potential to treat SR Ca2+ leak and consequently proarrhythmogenic events in rodent as well as in human atrial cardiomyocytes and cardiomyocytes from patients with heart failure. Further research is necessary towards clinical use in cardiac disease.

0265 : Tumour necrosis factor- carried by microparticles from apoptotic RAW 264.7 macrophage cells triggers deleterious effects on cardiomyocytes from adult mice

After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs) are part of the pathological process of unstable atherosclerotic plaques. In the present study, we have focused on MPs released by apoptotic murine RAW 264.7 macrophage cell line. The hypothesis that these MPs could induce deleterious effects in adult myocardial cells was evaluated. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α). Furthermore, cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to MPs. Conversely, Ca 2+ transient amplitudes and kinetics were not modified. The contractile effects of MPs were completely prevented after pre-treatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Moreover, confocal microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. Indeed, after 4 h of treatment with MPs, cardiomyocytes expressed increased apoptotic markers expression (caspase-3, caspase-8, Bax and cytochrome C) as demonstrated by Western Blot. The present study showed that these MPs carry functional TNF-α and activate TNFR1 in isolated adult murine ventricular cardiomyocytes, leading to cell shortening without affecting calcium signalling via a mechanism sensitive to NO synthase inhibitor and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. Thus, the present study allows supporting the hypothesis that MPs from human carotid atherosclerotic plaques may contain active TNF-α, which could contribute to MP-induced inflammatory signals in human atherosclerotic lesions leading to myocardial infarction. The author hereby declares no conflict of interest

Mitochondrial respiratory inhibition by 2,3-butanedione monoxime (BDM): implications for culturing isolated mouse ventricular cardiomyocytes.

Experiments in isolated ventricular cardiomyocytes have greatly facilitated the study of cellular and subcellular physiology in the heart. However, the isolation and culture of high-quality adult murine ventricular cardiomyocytes can be technically challenging. In most experimental protocols, the culture of viable adult murine cardiomyocytes for prolonged time periods is achieved with the addition of the myosin II ATPase inhibitors blebbistatin and/or 2,3-butanedione monoxime (BDM). These drugs are added to increase cell viability and life span by inhibiting spontaneous cardiomyocyte contraction, thereby preventing calcium overload, cell hypercontracture, and cell death. While the addition of BDM has been reported to prolong the life span of isolated adult murine cardiomyocytes, it is also associated with several off-target effects. Here, we report a novel off-target effect, in which BDM inhibits mitochondrial respiration by acting directly on the electron transport chain to reduce cell viability. In contrast, when cells were cultured with blebbistatin alone, cells survived for longer, and no metabolic off-target effects were observed. Based on these novel observations, we recommend that culture media for isolated mouse ventricular cardiomyocytes should be supplemented with blebbistatin alone, as BDM has the potential to affect mitochondrial respiration and cell viability, effects which may impact adversely on subsequent experiments.

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes.

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Abstract 182: Characterization of Electrophysiological and Conduction Parameters of the Heart in Trpm4-deficient Mice

TRPM4 is a Ca2+-activated non-selective cation channel that belongs to the family of the Transient Receptor Potential (TRP) ion channels. Importantly, TRPM4 is impermeable for Ca2+ and is involved in different Ca2+-dependent cell functions, such as exocytosis, contraction and cell death. Trpm4 is known to be expressed in atrial and ventricular cardiomyocytes. The interest in the functional role of TRPM4 in the heart has risen further by the discovery of Trpm4 mutations that are linked to cardiac conduction disorders, including Progressive Familial Heart Block type I (PFHBI) and Brugada Syndrome. Both gain-of-function and loss-of-function mutation were described in patients with cardiac conduction diseases. Recently, our group showed that TRPM4 plays a role during the late repolarization phase of the action potential in murine ventricular cardiomyocytes and that deletion of the Trpm4 gene leads to shorter ventricular action potentials. To characterize if deletion of Trpm4 has an effect on the conduction properties of the heart, an in depth electrophysiological study was performed in living mice. An octapolar catheter was inserted into the right atrium and ventricle of the heart to measure intracardial electrograms. The atrial-His (AH) and His-ventricular (HV) intervals were calculated and no differences were found between WT and Trpm4-deficient mice. Additionally, more detailed conduction parameters of the heart were determined by use of programmed electrical stimulation (PES) protocols. Sinus node recovery time (SNRT) was not different between WT and Trpm4-deficient mice. Effective refractory period of atrium (AERP), AV node (AVNERP) and ventricle (VERP) were in the same range in WT and Trpm4-deficient mice. Wenckebach periodicity, the parameter for AV nodal conduction, was also not different between WT and Trpm4-deficient mice. These results suggest that deletion of Trpm4 has no effects on the conduction properties of the murine heart.

Abstract 20451: Hedgehog Signaling and Cardiomyocyte Proliferation

Background: In contrast to adult mammalian heart, lower vertebrates such as newt exhibit a dramatic capacity for regeneration in response to injury. Understanding the underlying mechanisms and signaling pathways in newt could form the basis for regenerative therapies in mammals. In the present study, we explored the role of hedgehog (HH) signaling during cardiac regeneration. Methods and Results: To investigate cardiac regeneration (CR) in vivo, we performed ventricular resection studies in adult newt heart. Whole mount and histochemical studies revealed CR within 30 d of resection injury. Functional assessment by echocardiographic analysis showed improved ejection fraction from 22 ± 5% at 4 d to 42 ± 3% at 30 d of regeneration relative to control (60 ± 3%). Gene clustering and qRT-PCR analysis at 7d post injury indicated enrichment of hedgehog (HH) signaling factors including Shh and ptch1 by 3- and 2-fold respectively, suggesting a critical requirement of HH signaling at early stages of CR. We determined HH signaling is essential as pharmacological inhibition of the HH pathway resulted in complete ablation of CR. Using EdU-labeling and immunohistochemical analyses, we showed that HH signaling regulates proliferation of both epicardial and myocardial cells, as inhibition of the HH pathway reduced EdU-positive nuclei from 15 ± 2% to 5 ± 3%. In contrast, qRT-PCR analysis from murine hearts postnatal day (P) 2 to P14, showed reduced levels of HH signaling factors and cell-cycle associated mRNAs by 3.5-fold with concomitant increase of p21 by 2-fold. This implies HH signals are required for the proliferative process. Consistent with the newt studies, activation of HH in murine neonatal ventricular cardiomyocytes (NVCM) promoted cardiomyocyte proliferation, increasing positive nuclei from 10 ± 2% to 25 ± 3%, while inhibition of HH signaling reduced positivity to 6 ± 3%. qRT-PCR analysis established that activation of HH signaling in NVCM resulted in up regulation of transcripts associated with cardiomyocyte proliferation. Conclusion: These data indicated that HH signaling pathways modulate cardiomyocyte proliferation providing potential therapeutic targets for achieving mammalian cardiac regeneration.

Basal and β-Adrenergic Cardiomyocytes Contractility Dysfunction Induced by Dietary Protein Restriction is Associated with Downregulation of SERCA2a Expression and Disturbance of Endoplasmic Reticulum Ca2+ Regulation in Rats

Background: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. Methods: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca²⁺sparks analysis. Results: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca²⁺sparks were observed in PRG cardiomyocytes. Conclusion: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca²⁺ intracellular kinetics.

Abstract 48: Cardiac-Targeted TRPM7 Deletion Induces Cardiomyopathy, Heart Block and Impaired Ventricular Repolarization via Downregulation of Kcnd2 and Hcn4

Transient Receptor Potential Melastatin-7 (TRPM7) is a divalent permeant channel-kinase of unknown function. It is concentrated in myocardium during embryonic development, and TRPM7-like current has been reported in adult ventricular myocytes. In atrial cardiac fibroblasts, TRPM7 has been implicated in fibrogenesis in patients with atrial fibrillation, however its function in ventricular myocardium is unexplored. We tested the hypothesis that TRPM7 is required for ventricular function by crossing TRPM7fl/- mice with the cardiac-targeted αMHC-Cre line (αMHC-TRPM7 KO). We show that TRPM7 forms a functional current in adult murine ventricular cardiomyocytes and demonstrate that ∼50% of αMHC-TRPM7 KO mice develop a cardiomyopathy with left ventricular dysfunction and cardiac hypertrophy, associated with heart block and impaired ventricular repolarization. This phenotype is associated with robust elevations in mRNA of Postn, Nppa, Timp1, Acta and reductions in Hdac9, Kcnv2, Kcnj3, Kcnd2, Lgi1 and Hcn4. Consistent with mRNA expression analysis and the observed electrical remodeling, patch-clamp of αMHC-Cre-TRPM7 KO ventricular myocytes (RV and LV) reveals significant action potential prolongation (2.5 to 4-fold increase in APD50) due to a 2 to 4-fold reduction in transient outward current (Ito, encoded by Kcnd2). Similarly, the funny current (If), encoded by Hcn4, is diminished 4-fold in atrioventricular nodal cells isolated from αMHC-Cre-Trpm7 KO hearts, accounting for the observed heart block. The ∼50% of αMHC-Cre-Trpm7 KO mice that do not develop cardiomyopathy are devoid of heart block, show normal expression levels of hypertrophy genes Postn, Nppa, and Acta, but continue to show significant reductions in Kcnd2, Lgi1, Kcnj3, Kcnv2, and Hdac9 expression. In conclusion, these results demonstrate that cardiac-targeted TRPM7 deletion dysregulates the expression of ion channels important for cardiomyocyte repolarization (Kcnd2) and atrioventricular conduction (Hcn4), leading to impaired ventricular repolarization and heart block in mice. The observed cardiomyopathy may arise from hemodynamic stresses secondary to chronic bradycardia from heart block in the setting of down-regulated histone deacetylase 9 (HDAC9).

Cardiomyoplasty Improves Contractile Reserve after Myocardial Injury in Mice: Functional and Morphological Investigations with Reconstructive Three-Dimensional Echocardiography

Cellular cardiomyoplasty (CMP) is a novel therapeutic approach to myocardial injury (MI). Post-MI remodeling of the left ventricle (LV) comprises dilatation and impairment of systolic function and gives rise to progressive hemodynamic deterioration. We aimed to investigate: a) the impact of CMP on global and regional parameters of LV remodeling (LVR) as well as contractile reserve and b) the suitability and validity of different echocardiographic methods in this scenario. Murine ventricular cardiomyocytes (E13.5-E16.5) were transplanted into cryolesioned hearts of male HIM-OF1 mice. Echocardiography was performed at rest 4 and 14 days postoperatively. For quantification of akinetic myocardial mass and contractile reserve 2 weeks postoperatively additionally low-dose dobutamine stress echocardiography was conducted. Reconstructive 3D-echocardiography (r3D-echo) was compared to "plain" echocardiographic investigations and was compared to invasive measurements with conduction catheter. CMP significantly attenuated LV dilatation and reduced LV function decline on day 14, as obtained with all echocardiographic modalities and confirmed with conduction catheter measurements. In contrast to plain echocardiography and invasive testing, r3D-echo allowed noninvasive quantification of scar size and assessment of regional contractile reserve. Cell transplanted hearts demonstrated a significant decrease of akinetic myocardial mass (-CMP: 13 ± 2%; +CMP 7 ± 1%; p < 0.001) and increased regional contractile reserve, an indirect sign of myocardial viability. The present study demonstrates beneficial effects of CMP on global and regional parameters of LVR and contractile reserve after MI. In contrast to "simple" 2D echocardiography, r3D-echo allowed the assessment of regional contractile reserve and quantification of akinetic myocardial mass as additive functional and morphological measures of LVR.

Effect of MANT-nucleotides on L-type calcium currents in murine cardiomyocytes

Membranous adenylyl cyclases play a major role in G-protein-coupled receptor signalling and regulate various cellular responses, such as cardiac contraction. Cardiac apoptosis and development of cardiac dysfunction is prevented in mice lacking AC 5, a predominant isoform in the heart. In the search for a potent and selective AC 5 inhibitor, we recently identified 2'(3')-methylanthraniloyl-inosine-5'-triphosphate(MANT-ITP) as the most potent AC 5 inhibitor with a K ( i ) of 13 nM. Therefore, AC inhibition of MANT-ITP was assessed in ventricular cardiomyocytes and compared to three other MANT-nucleotides to evaluate its effect on cardiac signalling. Basal and isoproterenol-induced L-type calcium currents (I (Ca,L)) in murine ventricular cardiomyocytes were recorded by whole-cell patch-clamp technique, using four different MANT-nucleotides. The effects of the MANT-nucleotides on I (Ca,L) were unexpectedly complex. All MANT-nucleotides exhibited an inhibitory effect on basal I (Ca,L). Additionally, several MANT-nucleotides, i.e., MANT-ITPγS, MANT-ATP, and MANT-ITP, caused a strong initial increase in basal I (Ca,L) within the first 2.5 min that appeared to be unrelated to AC 5 inhibition. However, we detected a significant reduction on isoproterenol-induced I (Ca,L) with MANT-ITP, supporting the notion that AC 5 plays an important role in agonist-stimulated activation of I (Ca,L). Collectively, MANT-nucleotides are useful tools for the characterization of recombinant ACs, for fluorescence studies and crystallography, but in intact cardiomyocytes, caution must be exerted since MANT-nucleotides apparently possess additional effects than AC 5 inhibition, limiting their usefulness as tools for intact cell studies.

Angiotensin Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial Fibrosis and Diastolic Dysfunction

Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease. Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease.

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

Effect Of (−)-epigallocatechin Gallate (EGCG), A Green Tea Extract, On Excitation-contraction Coupling Of Murine Cardiomyocytes

Background: Polyphenolic compounds, green tea, reportedly have protective benefit for cardiovascular disease, but the mechanism(s) are unknown. We have recently found that green tea extracts such as (-)-epigallocatechin gallate(EGCG) bind to skeletal and cardiac ryanodine receptor(RyR) Ca2+-release channels. Here we examined the effect of EGCG(1μM, 100nM, 10nM) on cell shortening and Ca2+ kinetics in field-stimulated murine ventricular cardiomyocytes loaded with Fura-2AM and on cardiac RyR channels incorporated in lipid bilayers. Results: EGCG at 10 nM already maximally increased myocytes fractional shortening(%FS): ECGC 5.6±0.7, N=19, vs. Vehicle, 2.2±0.3, N=25, p 30-fold prolonging mean open time 15-fold without altering unitary conductance. EGCG did not alter SR Ca2+ loading capacity in the presence of RyR channel blocker ruthenium red. Taken together, these data suggest that EGCG enhances contractility of intact myocytes via its action on RyR channels. SR Ca2+ depletion is prevented by EGCG's concomitant inhibition of the NCX. Conclusions: EGCG potently modulates cardiac excitation contraction coupling by acting on RyR and possibly on L-type Ca2+-channel and NaCa exchanger. Supported by R01HL71670, R01HL88635, and R01AR43140

The novel adipocytokine visfatin exerts direct cardioprotective effects

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 × 10−6μmol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1 ± 4.1% in control hearts to 27.3 ± 4.0% (n≥ 6/group, P < 0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8 ± 5.5% and 45.9 ± 8.4%, respectively, versus 27.3 ± 4.0% with visfatin; n≥ 6/group, P < 0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2 ± 4.6% in control to 49.2 ± 3.7%(n > 200 cells/group, P < 0.05), an effect that was abrogated by wortmannin and U0126 (68.1 ± 5.2% and 59.7 ± 6.2%, respectively; n > 200 cells/group, P > 0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2 ± 4 sec. in control to 120 ± 7 sec. (n > 20 cells/group, P < 0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.

Enhancement of ventricular gap-junction coupling by rotigaptide.

Rotigaptide is proposed to exert its anti-arrhythmic effects by improving myocardial gap-junction communication. To directly investigate the mechanisms of rotigaptide action, we treated cultured neonatal murine ventricular cardiomyocytes with clinical pharmacological doses of rotigaptide and directly determined its effects on gap-junctional currents. Neonatal murine ventricular cardiomyocytes were enzymatically isolated and cultured for 1-4 days. Primary culture cell pairs were subjected to dual whole cell patch-clamp procedures to directly measure gap-junctional currents (I(j)) and voltage (V(j)). Rotigaptide (0-350 nM) was applied overnight or acutely perfused into 35 mm culture dishes. Rotigaptide (35-100 nM) acutely and chronically increased the resting gap-junction conductance (g(j)), and normalized steady-state minimum g(j) (G(min)) by 5-20%. Higher concentrations produced a diminishing response, which mimics the observed therapeutic efficacy of the drug. The inactivation kinetics was similarly slowed in a therapeutic concentration-dependent manner without affecting the V(j) dependence of inactivation or recovery. The effects of 0-100 nM rotigaptide on ventricular g(j) during cardiac action potential propagation were accurately modelled by computer simulations which demonstrate that clinically effective concentrations of rotigaptide can partially reverse conduction slowing due to decreases in g(j) and inactivation. These results demonstrate that therapeutic concentrations of rotigaptide increase the resting gap-junction conductance and reduce the magnitude and kinetics of steady-state inactivation in a concentration-dependent manner. Rotigaptide may be effective in treating re-entrant forms of cardiac arrhythmias by improving conduction and preventing the formation of re-entrant circuits in partially uncoupled myocardium.

Differential Myocardial Gene Delivery by Recombinant Serotype-Specific Adeno-associated Viral Vectors

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.

Cellular cardiomyoplasty improves survival after myocardial injury.

Cellular cardiomyoplasty is discussed as an alternative therapeutic approach to heart failure. To date, however, the functional characteristics of the transplanted cells, their contribution to heart function, and most importantly, the potential therapeutic benefit of this treatment remain unclear. Murine ventricular cardiomyocytes (E12.5-E15.5) labeled with enhanced green fluorescent protein (EGFP) were transplanted into the cryoinjured left ventricular walls of 2-month-old male mice. Ultrastructural analysis of the cryoinfarction showed a complete loss of cardiomyocytes within 2 days and fibrotic healing within 7 days after injury. Two weeks after operation, EGFP-positive cardiomyocytes were engrafted throughout the wall of the lesioned myocardium. Morphological studies showed differentiation and formation of intercellular contacts. Furthermore, electrophysiological experiments on isolated EGFP-positive cardiomyocytes showed time-dependent differentiation with postnatal ventricular action potentials and intact beta-adrenergic modulation. These findings were corroborated by Western blotting, in which accelerated differentiation of the transplanted cells was detected on the basis of a switch in troponin I isoforms. When contractility was tested in muscle strips and heart function was assessed by use of echocardiography, a significant improvement of force generation and heart function was seen. These findings were supported by a clear improvement of survival of mice in the cardiomyoplasty group when a large group of animals was analyzed (n=153). Transplanted embryonic cardiomyocytes engraft and display accelerated differentiation and intact cellular excitability. The present study demonstrates, as a proof of principle, that cellular cardiomyoplasty improves heart function and increases survival on myocardial injury.