Konrad Frank

Chronic Phospholamban–Sarcoplasmic Reticulum Calcium ATPase Interaction Is the Critical Calcium Cycling Defect in Dilated Cardiomyopathy

Dilated cardiomyopathy and end-stage heart failure result in multiple defects in cardiac excitation-contraction coupling. Via complementation of a genetically based mouse model of dilated cardiomyopathy, we now provide evidence that progressive chamber dilation and heart failure are dependent on a Ca2+ cycling defect in the cardiac sarcoplasmic reticulum. The ablation of a muscle-specific sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) inhibitor, phospholamban, rescued the spectrum of phenotypes that resemble human heart failure. Inhibition of phospholamban-SERCA2a interaction via in vivo expression of a phospholamban point mutant dominantly activated the contractility of ventricular muscle cells. Thus, interfering with phospholamban-SERCA2a interaction may provide a novel therapeutic approach for preventing the progression of dilated cardiomyopathy.

Evidence for Functional Relevance of an Enhanced Expression of the Na+-Ca2+ Exchanger in Failing Human Myocardium

BACKGROUND The present study aimed at investigating the expression of the Na(+)-Ca2+ exchanger and its functional role in human failing myocardium. METHODS AND RESULTS Na(+)-Ca2+ exchanger mRNA and protein levels were examined in nonfailing (NF, n = 8) and failing human myocardium (New York Heart Association functional class IV) with idiopathic dilated cardiomyopathy (DCM, n = 8) or ischemic heart disease (ICM, n = 6). The inotropic effect of the Na+ channel activator BDF 9148 was determined in electrically driven left ventricular papillary muscle strip preparations (1 Hz, 37 degrees C) from nonfailing (n = 8) and failing (n = 8) human hearts. Na(+)-Ca2+ exchanger mRNA levels were significantly increased, by 79% (P < .001) in DCM and by 58% (P < .01) in ICM compared with NF; protein levels increased by 36% (P < .001) and by 20% (P < .05), respectively. BDF 9148 increased the force of contraction concentration dependently, with a similar maximal effect in NYHA class IV and NF, but was more potent in NYHA class IV as demonstrated by a significantly smaller (P < .01) EC50 value (NYHA class IV, 0.18 [0.16 to 0.22] mumol/L; NF, 1.65 [1.3 to 3.0] mumol/L). In NYHA class IV, BDF 9148 (0.1 mumol/L) restored the positive force-frequency relationship and reduced the frequency-dependent increase in diastolic tension in relation to force of contraction. CONCLUSIONS The increased expression of the Na(+)-Ca2+ exchanger is a possible explanation for the increased inotropic potency of the Na+ channel activator BDF 9148 in failing human myocardium. The increase in exchanger molecules could be of functional relevance for the modulation of cardiac contractility by agents that increase the intracellular Na+ concentration. Enhancement of Na(+)-Ca2+ exchanger activity might be a powerful mechanism for increasing cardiac contractility in chronic heart failure.

Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation.

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.

Rescue of Contractile Parameters and Myocyte Hypertrophy in Calsequestrin Overexpressing Myocardium by Phospholamban Ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca2+-induced Ca2+ release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca2+ storage, assessed by caffeine-induced Na+-Ca2+ exchanger currents. The inactivation time of the L-type Ca2+ current (I Ca), which has an inverse correlation with Ca2+-induced SR Ca2+ release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability ofI Ca to trigger SR Ca2+ release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and α-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.

Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer

We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Modulation of SERCA: implications for the failing human heart.

Abstract Human heart failure is characterized by distinct alterations in the intracellular homeostasis and key regulators of the sarcoplasmic reticulum Ca2+ sequestration mechanisms. Systolic peak Ca2+ is reduced, diastolic Ca2+ levels are increased and diastolic Ca2+ decay is prolonged. Recently specific changes in the expression, function and modulation of SR Ca2+-ATPase (SERCA) have been elucidated. As such, in a variety of studies SERCA expression appeared to be decreased in the failing human heart, although these findings have been discussed controversially depending on the studied tissue, especially with respect to the non-failing samples and regional variation in the obtained samples. However, consistent findings of a diminished Ca2+ dependent SERCA activation were found. Increasing evidence has been provided that one of the underlying mechanisms for a decreased activation of SERCA is its altered regulation. With respect to this, the modulations through phospholamban and Ca2+-dependent protein kinase II (CaMK II) play a detrimental role in regulating SERCA function. Phospholamban phosphorylation of SERCA at the serine-16 and threonine-17 site is diminished in human heart failure resulting in decreases in the apparent affinity for Ca2+ of the SR Ca2+ uptake rates. In contrast, activation of CaMK II leads to an increased maximal velocity of SR Ca2+ sequestration that may enhance SR Ca2+-load. Additional regulation has been recently elucidated by changes in the apparent coupling ratio of Ca2+ transported per ATP hydrolysed. This review summarizes recent advances in the understanding how SERCA is modulated under physiological and pathophysiological conditions.

Phospholamban and cardiac contractility

Over the last three decades, our knowledge and understanding of the role of phospholamban and its modulation of sarcoplasmic reticulum (SR) function has advanced significantly. Phospholamban is a key regulator of cardiac contractility and modulates SR Ca2+ sequestration by inhibiting the SR Ca2+ -ATPase (SERCA) in its dephosphorylated state. Upon phosphorylation, which is mediated through β-adrenergic stimulation, the inhibitory effect of phospholamban on the function of SERCA is relieved. This review summarizes recent advances that have been made towards understanding the modulation of SR Ca2+-sequestration by phospholamban through the generation and characterization of genetically altered animal models. It also discusses the role of phospholamban in human heart failure and recent attempts to restore SR function in experimentally induced and human heart failure, which may be translated into future therapeutic approaches in the treatment of this disease.

Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure

OBJECTIVE Impaired myocyte Ca2+ handling is a common characteristic of failing hearts and increases in calcineurin activity, a Ca2+-sensitive phosphatase, have been implicated in heart failure phenotype. Transgenic mice with cardiac-specific expression of an active form of calcineurin display depressed function, hypertrophy and heart failure. We examined whether defects in cardiomyocyte Ca2+ handling properties contribute to the impaired cardiac function in calcineurin transgenic mice. METHODS The levels of SR Ca2+ handling proteins, SR Ca2+ transport function and cardiomyocyte mechanics, as well as Ca2+ kinetics were examined in mice overexpressing a constitutively active form of calcineurin. RESULTS Transgenic expression of activated calcineurin catalytic subunit resulted in significant protein increases (66%) in SERCA2 and decreases (35%) in phospholamban, as well as enhanced (approximately 80%) phospholamban phosphorylation. These alterations in the SR Ca2+-transport proteins resulted in increased V(max) and Ca2+-affinity of SERCA2. The myofibrillar Mg-ATPase activity was also significantly increased at pCa>6.0. The enhanced SR Ca2+ handling and Mg-ATPase activity reflected significant elevation in myocyte contractile parameters (3-fold), Ca2+ transient amplitude (1.5-fold) and the rate of Ca2+ signal decay (2-fold). In contrast, in vivo cardiac function assessed by echocardiography, indicated severely depressed contractility in calcineurin hearts. The apparent disparity in contractile properties between the cellular and multicellular preparations may be partially due to tissue remodeling, including interstitial fibrosis and a marked reduction (45%), dephosphorylation (81%) and redistribution of the gap junctional protein connexin-43, which could compromise intercellular communication. CONCLUSION Despite enhanced SR Ca2+ handling and contractility in myocytes, pathological remodeling and defects in intercellular coupling may underlie contractile dysfunction of the calcineurin hearts.

Crataegus special extract WS 1442 increases force of contraction in human myocardium cAMP-independently.

The mode of action of Crataegus extracts in the treatment of heart failure is still under examination. WS 1442, a standardized special extract from Crataegus leaves with flowers, exerts direct positive inotropic effects. This study was designed to investigate the mode of inotropic action of WS 1442 in human myocardium from patients with congestive heart failure (left ventricular myocardium from explanted hearts; NYHA IV, n = 8) as well as in nonfailing controls (right auricular trabeculae from patients with coronary heart disease, n = 8). WS 1442 effectively displaced specifically bound 3H-ouabain but did not influence the activity of adenylate cyclase [control, + Gpp(NH)p (10(-4) microM) 3,500 pmol cyclic adenosine monophosphate (cAMP)/20 min). In isolated left ventricular papillary muscle strips, WS 1442 significantly increased the force of contraction [basal, 1.8+/-0.2 mN; WS 1442 (50 microg/ml), 2.4+/-0.1 mN (130%)] and improved the frequency-dependent force generation (0.5 vs. 2.5 Hz: control, +0.1+/-0.01 mN; WS 1442, +0.9+/-0.3 mN) even in failing human myocardium. In fura-2-loaded muscle strips (right atrial trabeculae), WS 1442 increased both the Ca2+-transient and force generation. These effects also were observed in the lipophilic ethyl acetate-soluble fraction A, enriched in flavone derivatives. In conclusion, these findings suggest a pharmacologic mechanism of WS 1442 similar to the cAMP-independent positive inotropic action of cardiac glycosides. In addition, WS 1442 improves the force-frequency relation in failing human myocardium.