Kristin B. Andersson

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.

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.

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

BackgroundQuantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.ResultsThe abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.ConclusionsThe most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c‐Myb in vitro

In an attempt to elucidate signal transduction pathways which may modulate DNA binding of the transcription factor c‐Myb, we investigated whether c‐Myb could be a target for the signaling molecule nitric oxide (NO) in vitro. NO‐generating agents severely inhibited specific DNA binding of the c‐Myb minimal DNA‐binding domain R2R3. This inhibition was readily reversible upon treatment with excess DTT. A redox‐sensitive cysteine (C130) was required for this NO sensitivity. Moreover, a DNA‐binding domain carrying two of the avian myeloblastosis virus (AMV)‐specific mutations (L106H, V117D) appeared to be less sensitive to S‐nitrosylation than the wild‐type c‐Myb. This difference in NO sensitivity may influence the regulation of wild type versus AMV v‐Myb protein function.

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.

Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout

Cardiomyocyte contraction and relaxation are controlled by Ca2+ handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca2+ fluxes. Here we investigate whether altered Ca2+ fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na+/Ca2+-exchanger proximal to ryanodine receptors in the SR but did not contain Ca2+-channels. Ca2+ measurements demonstrated a switch from SR-driven to Ca2+ influx-driven Ca2+ transients in KO. Still, SR Ca2+ release constituted 20% of the Ca2+ transient in KO. Mathematical modeling suggested that Ca2+ influx via Na+/Ca2+-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca2+ release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.

Circulating cytokine levels in mice with heart failure are etiology dependent

OBJECTIVES The aim of this study was to examine whether alterations in circulating cytokine levels are dependent on the etiology of myocardial hypertrophy and heart failure (HF). BACKGROUND Several heart diseases are associated with altered levels of circulating cytokines. Cytokines are regarded as possible therapeutic targets or biomarkers, but such approaches are currently not in clinical use. If alterations in circulating cytokines are etiology dependent, this should be taken into consideration when using cytokines as disease markers and therapeutic targets. METHODS The serum levels of 25 cytokines were quantified with Luminex and/or ELISA in four murine models of heart disease: banding of the ascending aorta (AB) or the pulmonary artery (PB), myocardial infarction (MI), and a cardiomyopathy model with inducible cardiomyocyte-specific knockout of the sarco(endo)plasmatic reticulum Ca2+-ATPase (SERCA2KO). RESULTS No increase in circulating cytokine levels were found in mice 1 wk after AB, although substantial myocardial hypertrophy was present. After 1 wk of MI, only interleukin (IL)-18 was increased. In the SERCA2KO mice with HF, circulating levels of IL-1alpha, IL-2, IL-3, IL-6, IL-9, IL-10, IL-12p40, eotaxin, granulocyte-colony stimulating factor (G-CSF), interferon-gamma, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta were increased, and in mice with PB, IL-1alpha, IL-6, G-CSF, and monokine induced by gamma-interferon showed elevated levels. CONCLUSIONS Serum levels of cytokines in mice with HF vary depending on the etiology. Increased serum levels of several cytokines were found in models with increased right ventricular afterload, suggesting that the cytokine responses result primarily from systemic congestion.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP‐mediated processes, we have examined the expression of cAMP‐dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIα) was present in all the cells investigated, in contrast to the other R subunits (RIβ, RIIα, and RIIβ). Furthermore, three T cell lines and one B cell line expressed only RIα and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIα protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]‐thymidine incorporation and cell‐doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes. J. Cell. Physiol. 177:85–93, 1998.

Sequence Selectivity of c-Myb in Vivo RESOLUTION OF A DNA TARGET SPECIFICITY PARADOX

We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R2R3 in vitro and the more restricted preference of a R2R3VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1–360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1–360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R2R3 domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell.

Mice carrying a conditional Serca2flox allele for the generation of Ca2+ handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca(2+) into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2(null) mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2(flox) allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2(flox) mice with MLC-2v(wt/Cre) mice and with alphaMHC-Cre transgenic mice. In heterozygous Serca2(wt/flox)MLC-2v(wt/Cre) mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2(flox/flox) Tg(alphaMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2(flox) mouse is the first murine conditional gene disruption model for the SERCA family of Ca(2+) ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.