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Introduction: A basic prerequisite for a good surgical outcome in heart surgery is optimal myocardial protection. However, cardioplegia strategies used in adult cardiac surgery are not directly transferable to infant hearts. Paediatric microplegia, analogous to Calafiore cardioplegia used in adult cardiac surgery, offers the advantage of safe myocardial protection without haemodilution. The use of concentration-dependent paediatric microplegia is new in clinical implementation. Material and methods: Paediatric microplegia has been in clinical use in our institution since late 2014. It is applied via an 1/8 inch tube of a S5-HLM roller pump (LivaNova, Italy). As cardioplegic additive, a mixture of potassium (K) 20 mL (2 mmol/mL potassium chloride 14.9% Braun) and magnesium (Mg) 10 mL (4 mmol/mL Mg-sulphate Verla® i. v. 50%) is fixed into a syringe-pump (B. Braun, Germany). This additive is mixed with arterial patient blood from the oxygenator in different flow dependent ratios to form an effective cardioplegia. Technique: After microplegia application of initially 25 mmol/L K with 11 mmol/L Mg for 2 min, a safe cardioplegic cardiac arrest is achieved, which after release of the coronary circulation, immediately returns to a spontaneous cardiac-rhythm. In the case of prolonged aortic clamping, microplegia is repeated every 20 min with a reduction of the application dose of K by 20% and Mg by 30% (20 mmol/L K; 8.5 mmol/L Mg) and a further reduction down to a maintenance dose (15 mmol/L K; 6 mmol/L Mg) after additional 20 min. Summary: The microplegia adapted to the needs of paediatric myocardium is convincing due to its simple technical implementation for the perfusionist while avoiding haemodilution. However, the required intraoperative interval of microplegia of approx. 20 min demands adapted intraoperative management from the surgeon. [ABSTRACT FROM AUTHOR]

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death. [ABSTRACT FROM AUTHOR]

The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaL directly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+ channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaL in different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaL density by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of total cacna1c and of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaL density by 30%, mildly slowed ICaL inactivation and shifted steady-state inactivation to more negative potentials. Total cacna1c mRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaL density and cacna1c mRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Organotypic culture of human ventricular myocardium is emerging in basic and translational cardiac research. However, few institutions have access to human ventricular tissue, whereas atrial tissue is more commonly available and important for studying atrial physiology. This study presents a method for long-term cultivation of beating human atrial myocardium. After written informed consent, tissues from the right-atrial appendage were obtained from patients with sinus rhythm undergoing open heart surgery with cardiopulmonary bypass. Trabeculae (pectinate muscles) prepared from the samples were installed into cultivation chambers at 37°C with a diastolic preload of 500 μN. After 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz. Contractile force was monitored continuously. Beta-adrenergic response, refractory period (RP) and maximum captured frequency (fmax) were assessed periodically. After cultivation, viability and electromechanical function were investigated, as well as the expression of several genes important for intracellular Ca2+ cycling and electrophysiology. Tissue microstructure was analyzed by confocal microscopy. We cultivated 19 constantly beating trabeculae from 8 patient samples for 12 days and 4 trabeculae from 3 specimen for 21 days. Functional parameters were compared directly after installation (0 d) with those after 12 d in culture. Contraction force was 384 ± 69 μN at 0 d and 255 ± 90 μN at 12 d (p = 0.8, n = 22), RP 480 ± 97 ms and 408 ± 78 ms (p = 0.3, n = 9), fmax 3.0 ± 0.5 Hz and 3.8 ± 0.5 Hz (p = 0.18, n = 9), respectively. Application of 100 nM isoprenaline to 11 trabeculae at 7 d increased contraction force from 168 ± 35 μN to 361 ± 60 μN (p < 0.01), fmax from 6.4 ± 0.6 Hz to 8.5 ± 0.4 Hz (p < 0.01) and lowered RP from 319 ± 22 ms to 223 ± 15 ms. CACNA1c (L-type Ca2+ channel subunit) and GJA1 (connexin-43) mRNA expressions were not significantly altered at 12 d vs 0 d, while ATP2A (SERCA) and KCNJ4 (Kir2.3) were downregulated, and KCNJ2 (Kir2.1) was upregulated. Simultaneous Ca2+ imaging and force recording showed preserved excitation-contraction coupling in cultivated trabeculae. Confocal microscopy indicated preserved cardiomyocyte structure, unaltered amounts of extracellular matrix and gap junctions. MTT assays confirmed viability at 12 d. We established a workflow that allows for stable cultivation and functional analysis of beating human atrial myocardium for up to 3 weeks. This method may lead to novel insights into the physiology and pathophysiology of human atrial myocardium. [ABSTRACT FROM AUTHOR]

Cryopreservation of whole organs and specific tissues is an important and continually expanding field of medicine. The protocols currently used for organ preservation do not ensure survivability and functionality; the protocols for ovarian tissue lead to acceptable outcomes, but these are still capable of further improvement. In general, cryopreservation protocols need to be optimized. One important approach to improving cryopreservation protocols in general involves reducing exposure to cytotoxic cryoprotective agents prior to freezing. This study, therefore, evaluated the real-time tissue penetration of dimethyl sulfoxide, a cryoprotective agent that is widely used in cryopreservation. Dimethyl sulfoxide penetration in rat hearts perfused with a 15% (v/v) dimethyl sulfoxide solution was examined in real-time using dynamic contrast-enhanced micro-computed tomography imaging. Viability of cardiomyocytes was not significantly affected by the dimethyl sulfoxide perfusion procedure. Two different perfusion rates were evaluated and compared with perfusion using a common iodine-based contrast agent (iomeprol). The dynamic contrast-enhanced micro-computed tomography imaging data showed that dimethyl sulfoxide flushes both the extracellular and intracellular spaces in rat heart tissue to 95% equilibration after ≈ 35 s via perfusion. Subsequent wash-out via perfusion is completed to 95% within ≈ 49 s. The equilibration duration routinely used in dimethyl sulfoxide–based protocols for cryopreservation should therefore be questioned. Shorter incubation duration would perhaps be sufficient, as well as being beneficial in relation to cell survivability. It would be helpful to have techniques for non-invasive real-time monitoring of the penetration of cryoprotective agents and such techniques should be used to revise cryopreservation protocols. Switching to perfusion-based equilibration procedures might be beneficial, if feasible. [ABSTRACT FROM AUTHOR]

The α1-adrenoceptor as well as the AT1- and the ETA-receptor couple to G-proteins of the Gαq/11 family and contribute to the regulation of the transient outward K+ current (Ito,f) under pathological conditions such as cardiac hypertrophy or failure. This suggests an important role of Gαq/11-signalling in the physiological regulation of Ito,f. Here, we investigate mice deficient of the Gα11 protein (gna11−/−) to clarify the physiological role of Gα11 signalling in cardiac ion channel regulation. Myocytes from endocardial and epicardial layers were isolated from the left ventricular free wall and investigated using the ruptured-patch whole-cell patch-clamp technique. At +40 mV, epicardial myocytes from gna11−/− mice displayed a 23% larger Ito,f than controls (52.6 ± 4.1 pApF−1, n = 20 vs 42.7 ± 2.8 pApF−1, n = 26, p < 0.05). Endocardial Ito,f was similar in gna11−/− mice and controls. With the except of minor changes in endocardial myocytes, Ito,f kinetics were similar in both groups. In the epicardial layer, western blot analysis revealed a 19% higher expression of the K+-channel α-subunit Kv4.2 in gna11−/− mice than in wild type (wt; p < 0.05). The β-subunit KChIP2b was upregulated by 102% in epicardial myocytes of gna11−/− mice ( p < 0.01, n = 4). Consistent with the difference in Ito,f, action potential duration was shorter in epicardial cells of gna11−/− mice than in wt ( p < 0.05), while no difference was found in endocardial myocytes. These results suggest that Gα11-coupled signalling is a central pathway in the regulation of Ito,f. It physiologically exerts a tonic inhibitory influence on the expression of Ito,f and thereby contributes to the regulation of cardiac repolarisation. [ABSTRACT FROM AUTHOR]

Mineralocorticoid receptor (MR) activation modulates cardiac L-type Ca2+ current ( I CaL) and transient outward K+ current ( I to). The exact circumstances of MR activation, however, remain elusive. Here, we investigate the influence of corticosteroids on MR-mediated changes in cellular electrophysiology. In vitro incubation of adult rat ventricular myocytes with the MR agonist aldosterone (100 nM, 24 h) increased I CaL density by 34% ( n = 16; p < 0.01). This effect was abrogated by co-incubation with the MR antagonist spironolactone (10 μM). To investigate whether an increase in serum aldosterone concentration is sufficient for an increase in I CaL in vivo, rats were subjected to low Na+ diet (LSD, 0.013% Na+) for 28 days. This increased serum aldosterone concentration from 0.19 ± 0.04 nM ( n = 6) in control animals (0.3% Na+, CSD) to 16.1 ± 2.1 nM ( n = 6; p < 0.0001). Strikingly, I CaL density was similar in both CSD and LSD rats (−12.9 ± 0.9 pA pF−1, n = 18 and −13.7 ± 1.1 pA pF−1, n = 16, respectively), as was I to density. In vitro, the glucocorticoid corticosterone (1 μM) also increased I CaL and this effect was blocked by spironolactone (10 μM). Co-incubation with corticosterone (1 μM, the normal serum concentration) and aldosterone (100 nM, mimicking low Na+ intake) did not further increase I CaL compared to corticosterone alone. Moreover, co-incubation of myocytes with N-acetylcysteine (10 mM) prevented the aldosterone (100 nM) or corticosterone (1 μM)-induced increase in I CaL. In conclusion, an increase in serum aldosterone concentration in response to LSD is not sufficient for an increase in I CaL density in cardiomyocytes in vivo. This is supported in vitro by the absence of an effect of aldosterone on I CaL in the presence of a physiological concentration of corticosterone. Moreover, the cellular redox state may modulate MR activation. [ABSTRACT FROM AUTHOR]

Inhibition of endothelin-A (ETA) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ETA-receptor inhibition on the development of regional alterations of the transient outward K+ current ( I to) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague–Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ETA-receptor antagonist darusentan (LU135252, 35 mg [kg body weight]−1 day−1) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight ( p < 0.001) and caused a significant reduction in I to (at +40 mV) in epicardial myocytes (19.5 ± 1.2 pA pF−1, n = 32 vs 23.2 ± 1.2 pA pF−1, n = 35, p < 0.05). Darusentan further reduced I to in AS (15.4 ± 1.3 pA pF−1, n = 37, p < 0.05) and sham-operated animals (19.8 ± 1.6 pA pF−1, n = 48, ns.). The effects of AS and darusentan on I to were significant and independent as tested by two-way analysis of variance. I to was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I to in the epicardial region of the left ventricle but that ETA-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: A reduction of the Ca2+-independent transient outward potassium current (I to) in epicardial but not in endocardial myocytes of the left ventricle has been observed in cardiac hypertrophy and is thought to contribute to the electrical vulnerability associated with this pathology. Methods: In the present study we investigated the molecular mechanisms underlying regional alterations in I to in hypertrophied hearts of spontaneously hypertensive rats (SHR) using the whole-cell patch-clamp technique, quantitative RT-PCR and heterologous expression of underlying ion channel subunits. Results: I to was significantly smaller in epicardial myocytes of SHR than in Wistar-Kyoto (WKY) controls (11.1±0.9 pA/pF, n =20 vs. 16.8±1.7 pA/pF, n =20, p <0.01), but not different in endocardial myocytes from both groups. Quantitative RT-PCR analysis of the genes encoding I to revealed significantly lower levels of Kv4.2 and Kv4.3 mRNA in the epicardial region of SHR rats compared to WKY rats. In contrast, mRNA expression levels of all three splice variants of the β-subunit KChIP2 were significantly higher in both endo- and epicardial myocytes from SHR than from WKY rats. In parallel, inactivation of I to, which is negatively modulated by KChIP2, was slowed down in SHR while recovery from inactivation remained unchanged. Heterologous co-expression of increasing amounts of KChIP2b together with a fixed amount of Kv4.2 in Xenopus laevis oocytes revealed a hyperbolic relation of recovery from inactivation and inactivation time constant, demonstrating that KChIP2 preferentially affects inactivation, if its expression level is high. Conclusion: These results suggest that downregulation of I to in the left ventricle of SHR is mediated by a reduced expression of Kv4.2 and Kv4.3 (but not of KChIP2), whereas the slower inactivation of I to can be explained by increased expression levels of KChIP2 in SHR. [Copyright &y& Elsevier]

Background/Aims: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K+ channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pHe); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pHe on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. Results: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pHe. hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pHe: The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. Conclusion: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A decrease of the transient outward potassium current (Ito) has been observed in cardiac hypertrophy and contributes to the altered shape of the action potential (AP) of hypertrophied ventricular myocytes. Since the shape and duration of the ventricular AP are important determinants of the Ca2+ influx during the AP (QCa), we investigated the effect of ascending aortic stenosis (AS) on QCa in endo- and epicardial myocytes of the left ventricular free wall using the AP voltage-damp technique. In sham-operated animals, QCa was significantly larger in endocardial compared to epicardial myocytes (803 ± 65 fC pF-1, n = 27 vs. 167 ± 32 fC pF-1, n = 38, P < 0.001). Ascending aortic stenosis significantly increased QCa in epicardial myocytes (368 ± 54 fC pF-1, n = 42, P < 0.05), but did not alter QCa in endocardial myocytes (696 ± 65 fC pF-1, n = 26). Peak and current-voltage relation of the AP-induced Ca2+ current were unaffected by AS. However, the time course of the current-voltage relation was significantly prolonged in epicardial myocytes of AS animals. Model calculations revealed that the increase in QCa can be ascribed to a prolonged opening of the activation gate, whereas an increase in inactivation prevents an excessive increase in QCa. In conclusion, AS significantly increased AP-induced Ca2+ influx in epicardial but not in endocardial myocytes of the rat left ventricle. [ABSTRACT FROM AUTHOR]

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddle’s syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na+. We tested the effect of extracellular Na+ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na+ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddle’s syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na+ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na+ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddle’s syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na+ concentration [Na+]i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na+]i-app. We conclude that the inhibitory effect of extracellular Na+ removal on rundown is due to an inhibition of endocytic ENaC retrieval. [ABSTRACT FROM AUTHOR]

Objective: Several members of the PKD gene family (PKD2, PKDL and PKD2L2) are expressed in the heart. Polycystin-2 and its homologues, which are encoded by these genes, have recently been shown to form Ca2+-regulated nonselective cation channels in heterologous expression systems. Previously, large conductance nonselective cation channels (LCC) have been described in cardiomyocytes, however, their molecular identity remained obscure. We therefore examined whether LCCs may be formed by polycystins. Methods: Myocytes isolated from the rat left ventricle were investigated by the whole-cell patch-clamp technique and single-cell RT-PCR. Results: Application of 10 mM caffeine to the bath solution to increase the intracellular Ca2+ concentration led to activation of LCC in 56% of the myocytes investigated (total n=651), in ∼10%, more than three LCCs were detected. The single channel conductance was ∼300 pS for monovalent cations and the channel was relatively nonselective for the monovalent cations Na+, K+, Li+, and Cs+ and also permeable for the divalent cations Ca2+ and Ba2+, but impermeable for NMDG+ and Cl−. Amiloride (IC50=131±1.1 μM) and millimolar concentrations of the trivalent cations Gd3+ and La3+ inhibited the LCC. Single-cell RT-PCR analysis revealed that mRNA of PKD2 and PKD2L2, but not PKDL or PKD1 are expressed in individual rat left ventricular myocytes. Conclusion: The characteristics of LCC shown in the present study are nearly identical to those observed for polycystin-2 and its homologues suggesting that polycystin-2 or polycystin-2L2 underlie LCC in ventricular myocytes. [Copyright &y& Elsevier]

Cardiac hypertrophy alters the regional distribution of action potential duration by affecting the magnitude and kinetics of underlying ionic currents. In the present study, the effect of pressure-induced cardiac hypertrophy on the magnitude and kinetics of the L-type Ca2+ current (ICaL) was investigated in endo- and epicardial myocytes of the rat left ventricular free wall. Cardiac hypertrophy was induced by stenosis of the ascending aorta (AS), which led to a pressure difference between the left ventricular peak pressure and the peripheral systolic blood pressure of 84±6 mmHg (n=5, P<0.0001). Sham-operated animals served as controls (n=7). ICaL was investigated using the whole-cell patch-clamp technique. The magnitude of ICaL as well as its inactivation, steady-state activation, steady-state inactivation and recovery from inactivation were similar in endo- and epicardial myocytes of sham-operated rats. In myocytes isolated from AS animals, ICaL magnitude was significantly greater than in sham-operated animals (2407±127 pA, n=42 versus 1904±102 pA, n=33; P<0.01). This difference disappeared, however, when ICaL was normalized to cell capacitance. The kinetic properties of ICaL were unaffected by AS in both endo- and epicardial myocytes. In conclusion, in pressure-induced cardiac hypertrophy the density and kinetics of ICaL are preserved. This suggests that an additional insertion or recruitment of channels normally underlying ICaL into the cell membrane compensates for the increased cell size associated with hypertrophy. [ABSTRACT FROM AUTHOR]

Chronic heart failure (HF) in adults causes remodeling of the cardiomyocyte transverse tubular system (t-system), which contributes to disease progression by impairing excitation-contraction (EC) coupling. However, it is unknown if t-system remodeling occurs in pediatric heart failure. This study investigated the t-system in pediatric viral myocarditis. The t-system and integrity of EC coupling junctions (co-localization of L-type Ca 2+ channels with ryanodine receptors and junctophilin-2) were analyzed by 3D confocal microscopy in left-ventricular (LV) samples from 5 children with myocarditis (age 14 ± 3 months), undergoing ventricular assist device (VAD) implantation, and 5 children with atrioventricular septum defect (AVSD, age 17 ± 3 months), undergoing corrective surgery. LV ejection fraction (EF) was 58.4 ± 2.3% in AVSD and 12.2 ± 2.4% in acute myocarditis. Cardiomyocytes from myocarditis samples showed increased t-tubule distance (1.27 ± 0.05 μm, n = 34 cells) and dilation of t-tubules (volume-length ratio: 0.64 ± 0.02 μm 2 ) when compared with AVSD (0.90 ± 0.02 μm, p < 0.001; 0.52 ± 0.02 μm 2 , n = 61, p < 0.01). Intriguingly, 4 out of 5 myocarditis samples exhibited sheet-like t-tubules (t-sheets), a characteristic feature of adult chronic heart failure. The fraction of extracellular matrix was slightly higher in myocarditis (26.6 ± 1.4%) than in AVSD samples (24.4 ± 0.8%, p < 0.05). In one case of myocarditis, a second biopsy was taken and analyzed at VAD explantation after extensive cardiac recovery (EF from 7 to 56%) and clinical remission. When compared with pre-VAD, t-tubule distance and density were unchanged, as well as volume-length ratio (0.67 ± 0.04 μm 2 vs. 0.72 ± 0.05 μm 2 , p = 0.5), reflecting extant t-sheets. However, junctophilin-2 cluster density was considerably higher (0.12 ± 0.02 μm -3 vs. 0.05 ± 0.01 μm -3 , n = 9/10, p < 0.001), approaching values of AVSD (0.13 ± 0.05 μm -3 , n = 56), and the measure of intact EC coupling junctions showed a distinct increase (20.2 ± 5.0% vs. 6.8 ± 2.2%, p < 0.001). Severe t-system loss and remodeling to t-sheets can occur in acute HF in young children, resembling the structural changes of chronically failing adult hearts. T-system remodeling might contribute to cardiac dysfunction in viral myocarditis. Although t-system recovery remains elusive, recovery of EC coupling junctions may be possible and deserves further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fiegle, Schöber, Dittrich, Cesnjevar, Klingel, Volk, Alkassar and Seidel.)

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 µm thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 ± 11.9 vs. 7.89 ± 3.6%, p < 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 ± 15.0 vs. 6.87 ± 5.23 cells/mg, p < 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9-200 cells/mg) from minimal amounts of tissue (<50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD 90 ), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2-2 Hz pacing frequency. F 1 Hz /F 0 . 5 Hz and F 2 Hz /F 0 . 5 Hz served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F 1 Hz /F 0 . 5 Hz ( R 2 = 0.82) and F 2 Hz /F 0 . 5 Hz ( R 2 = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F 1 Hz /F 0 . 5 Hz , but to a lesser degree ( R 2 = 0.49), whereas NCX1 was not correlated ( R 2 = 0.02). CD 90 correlated positively with ΔTT ( R 2 = 0.39) and negatively with SERCA2/PLB ( R 2 = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP ( R 2 = 0) but rather with TTR ( R 2 = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model ( R 2 = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation., (Copyright © 2020 Abu-Khousa, Fiegle, Sommer, Minabari, Milting, Heim, Weyand, Tomasi, Dendorfer, Volk and Seidel.)

Background: Cardiovascular resuscitation upon intoxication with lipophilic ion channel-blocking agents has proven most difficult. Recently, favorable results have been reported when lipid rescue therapy is performed, i.e., the infusion of a triglyceride-rich lipid emulsion during resuscitation. However, the mechanism of action is poorly understood., Methods: The authors investigate the effects of a clinically used lipid emulsion (Lipovenös® MCT 20%; Fresenius Kabi AG, Bad Homburg, Germany) on the block of the fast Na current (INa) induced by the lipophilic local anesthetic bupivacaine in adult rat left ventricular myocytes by using the whole cell patch clamp technique., Results: Bupivacaine at 10 µm decreased INa by 54% (-19.3 ± 1.9 pApF vs. -42.3 ± 4.3 pApF; n = 17; P < 0.001; VPip = -40 mV, 1 Hz). Addition of 10% lipid emulsion in the presence of bupivacaine produced a 37% increase in INa (-26.4 ± 2.8 pApF; n = 17; P < 0.001 vs. bupivacaine alone). To test whether these results could be explained by a reduction in the free bupivacaine concentration by the lipid (lipid-sink effect), the authors removed the lipid phase from the bupivacaine-lipid mixture by ultracentrifugation. Also, the resulting water phase led to an increase in INa (+19%; n = 17; P < 0.001 vs. bupivacaine), demonstrating that part of the bupivacaine had been removed during ultracentrifugation. The substantially less lipophilic mepivacaine (40 µm) reduced INa by 27% (n = 24; P < 0.001). The mepivacaine-lipid mixture caused a significant increase in INa (+17%; n = 24; P < 0.001). For mepivacaine, only a small lipid-sink effect could be demonstrated (+8%; n = 23; P < 0.01), reflecting its poor lipid solubility., Conclusion: The authors demonstrate lipid rescue on the single-cell level and provide evidence for a lipid-sink mechanism.

BNP (brain-type natriuretic peptide) is a cardiac hormone with systemic haemodynamic effects as well as local cytoprotective and antiproliferative properties. It is induced under a variety of pathophysiological conditions, including decompensated heart failure and myocardial infarction. Since regional hypoxia is a potential common denominator of increased wall stretch and myocardial hypoperfusion, we investigated the direct effects of hypoxia on BNP expression, and the role of the HIF (hypoxia-inducible transcription factor) in BNP regulation. Using an RNase protection assay we found a strong hypoxic induction of BNP mRNA expression in different cell lines and in cultured adult rat cardiomyocytes. Systemic hypoxia and exposure to 0.1% CO induced BNP expression in the rodent myocardium in vivo, although this was at a lower amplitude. BNP promoter-driven luciferase expression increased 10-fold after hypoxic stimulation in transient transfections. Inactivation of four putative HREs (hypoxia-response elements) in the promoter by site-directed mutagenesis revealed that the HRE at -466 nt was responsible for hypoxic promoter activation. A functional CACAG motif was identified upstream of this HRE. The HIF-1 complex bound specifically and inducibly only to the HRE at -466 nt, as shown by EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation). siRNA (small interfering RNA)-mediated knockdown of HIF-1alpha, but not HIF-2alpha, interfered with hypoxic BNP mRNA induction and BNP promoter activation, confirming that BNP is a specific HIF-1alpha target gene. In conclusion, BNP appears to be part of the protective program steered by HIF-1 in response to oxygen deprivation. Induction of BNP may therefore contribute to the potential benefits of pharmacological HIF inducers in the treatment of ischaemic heart disease and heart failure.