William E. Louch

Methods in cardiomyocyte isolation, culture, and gene transfer.

Since techniques for cardiomyocyte isolation were first developed 35 years ago, experiments on single myocytes have yielded great insight into their cellular and sub-cellular physiology. These studies have employed a broad range of techniques including electrophysiology, calcium imaging, cell mechanics, immunohistochemistry and protein biochemistry. More recently, techniques for cardiomyocyte culture have gained additional importance with the advent of gene transfer technology. While such studies require a high quality cardiomyocyte population, successful cell isolation and maintenance during culture remain challenging. In this review, we describe methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart. This discussion outlines general principles for the beginner, but also provides detailed specific protocols and advice for common caveats. We additionally review methods for short-term myocyte culture, with particular attention given to the importance of substrate and media selection, and describe time-dependent alterations in myocyte physiology that should be anticipated. Gene transfer techniques for neonatal and adult cardiomyocytes are also reviewed, including methods for transfection (liposome, electroporation) and viral-based gene delivery.

T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction

In cardiac myocytes, initiation of excitation–contraction coupling is highly localized near the T‐tubule network. Myocytes with a dense T‐tubule network exhibit rapid and homogeneous sarcoplasmic reticulum (SR) Ca2+ release throughout the cell. We examined whether progressive changes in T‐tubule organization and Ca2+ release synchrony occur in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by ligation of the left coronary artery, and CHF was diagnosed by echocardiography (left atrial diameter >2.0 mm). CHF mice were killed at 1 or 3 weeks following MI (1‐week CHF, 3‐week CHF) and cardiomyocytes were isolated from viable regions of the septum, excluding the MI border zone. Septal myocytes from SHAM‐operated mice served as controls. T‐tubules were visualized by confocal microscopy in cells stained with di‐8‐ANEPPS. SHAM cells exhibited a regular striated T‐tubule pattern. However, 1‐week CHF cells showed slightly disorganized T‐tubule structure, and more profound disorganization occurred in 3‐week CHF with irregular gaps between adjacent T‐tubules. Line‐scan images of Ca2+ transients (fluo‐4 AM, 1 Hz) showed that regions of delayed Ca2+ release occurred at these gaps. Three‐week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca2+ release. A common pattern of Ca2+ release in 3‐week CHF was maintained between consecutive transients, and was not altered by forskolin application. Thus, progressive T‐tubule disorganization during CHF promotes dyssynchrony of SR Ca2+ release which may contribute to the slowing of SR Ca2+ release in this condition.

Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca(2+) from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca(2+) necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca(2+) content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca(2+) transients were predominantly generated by enhanced Ca(2+) flux through L-type Ca(2+) channels and the Na(+)-Ca(2+) exchanger. Moreover, equivalent increases in cytosolic [Ca(2+)] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca(2+) transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase α2-isoform in heart failure

AIMS The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout

Alterations in trans‐sarcolemmal and sarcoplasmic reticulum (SR) Ca2+ fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte‐specific excision of the SR Ca2+‐ATPase (SERCA) gene. At 4 weeks following gene deletion (4‐week KO) cardiac function remained near normal values. However, end‐stage heart failure developed by 7 weeks (7‐week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4‐ and 7‐week KO, and relaxation was slowed. Ca2+ transients were similarly altered. Reduction in Ca2+ transient magnitude resulted from complete loss of SR Ca2+ release between 4‐ and 7‐week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca2+ release was partly offset by increased L‐type Ca2+ current, which was facilitated by AP prolongation in 7‐week KO. Ca2+ entry via reverse‐mode Na+–Ca2+ exchange (NCX) was also enhanced. Up‐regulation of NCX and plasma membrane Ca2+‐ATPase increased Ca2+ extrusion rates in 4‐week KO. Diastolic dysfunction in 7‐week KO resulted from further SERCA2 loss, but also impaired NCX‐mediated Ca2+ extrusion following Na+ accumulation. Reduced Na+‐K+‐ATPase activity contributed to the Na+ gain. Normalizing [Na+] by dialysis increased the Ca2+ decline rate in 7‐week KO beyond 4‐week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na+ accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na+ gain is prevented, up‐regulated Ca2+ extrusion mechanisms can maintain near‐normal diastolic function in the absence of SERCA2.

There Goes the Neighborhood: Pathological Alterations in T-Tubule Morphology and Consequences for Cardiomyocyte Ca2+ Handling

T-tubules are invaginations of the cardiomyocyte membrane into the cell interior which form a tortuous network. T-tubules provide proximity between the electrically excitable cell membrane and the sarcoplasmic reticulum, the main intracellular Ca2+ store. Tight coupling between the rapidly spreading action potential and Ca2+ release units in the SR membrane ensures synchronous Ca2+ release throughout the cardiomyocyte. This is a requirement for rapid and powerful contraction. In recent years, it has become clear that T-tubule structure and composition are altered in several pathological states which may importantly contribute to contractile defects in these conditions. In this review, we describe the “neighborhood” of proteins in the dyadic cleft which locally controls cardiomyocyte Ca2+ homeostasis and how alterations in T-tubule structure and composition may alter this neighborhood during heart failure, atrial fibrillation, and diabetic cardiomyopathy. Based on this evidence, we propose that T-tubules have the potential to serve as novel therapeutic targets.

A dominant STIM1 mutation causes Stormorken syndrome.

Stormorken syndrome is a rare autosomal‐dominant disease with mild bleeding tendency, thrombocytopathy, thrombocytopenia, mild anemia, asplenia, tubular aggregate myopathy, miosis, headache, and ichthyosis. A heterozygous missense mutation in STIM1 exon 7 (c.910C>T; p.Arg304Trp) (NM_003156.3) was found to segregate with the disease in six Stormorken syndrome patients in four families. Upon sensing Ca2+ depletion in the endoplasmic reticulum lumen, STIM1 undergoes a conformational change enabling it to interact with and open ORAI1, a Ca2+ release‐activated Ca2+ channel located in the plasma membrane. The STIM1 mutation found in Stormorken syndrome patients is located in the coiled‐coil 1 domain, which might play a role in keeping STIM1 inactive. In agreement with a possible gain‐of‐function mutation in STIM1, blood platelets from patients were in a preactivated state with high exposure of aminophospholipids on the outer surface of the plasma membrane. Resting Ca2+ levels were elevated in platelets from the patients compared with controls, and store‐operated Ca2+ entry was markedly attenuated, further supporting constitutive activity of STIM1 and ORAI1. Thus, our data are compatible with a near‐maximal activation of STIM1 in Stormorken syndrome patients. We conclude that the heterozygous mutation c.910C>T causes the complex phenotype that defines this syndrome.

An analysis of deformation‐dependent electromechanical coupling in the mouse heart

•  The amount of force generated by heart cells is strongly influenced by feedback from the deformation of cardiac tissue, both from the changes in cell length and the rate at which cells are stretched. •  We analysed the effect these cellular mechanisms have on whole heart function by making a computational model of mouse heart cells, and embedding this cellular model into a representation of the heart. •  Unlike previous murine models, this model represents the heart at both body temperature and the high heart rates seen in these animals, allowing us to directly compare results from our computational model with experimental measurements. •  Results show that effects from the rate of stretch are especially important for explaining the large differences observed between force generated by isolated cells and pressure measured experimentally. •  The model also provides an important framework for future research focused on interpreting results from genetic manipulation experiments in mice.

Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes

AIMS To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development. METHODS AND RESULTS SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results). CONCLUSION Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

Syndecan-4 Is Essential for Development of Concentric Myocardial Hypertrophy via Stretch-Induced Activation of the Calcineurin-NFAT Pathway

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4−/− mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4−/−-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.