Distinct interactions between actin and essential myosin light chain isoforms.

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.

Spinophilin is required for normal morphology, Ca(2+) homeostasis and contraction but dispensable for ß-adrenergic stimulation of adult cardiomyocytes.

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and ß-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) ß-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for ß-adrenergic stimulation of adult cardiomyocytes.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity.

AIMS: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac ß-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or ß-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rß-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. CONCLUSION: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

Auto-inhibitory effects of an IQ motif on protein structure and function.

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH(664-915) was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH(664-915). Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of I814, Q815, R819, and W827 stretched MYH(664-915) and increased 30-fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin.

Minigenes encoding N-terminal domains of human cardiac myosin light chain-1 improve heart function of transgenic rats.

In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart.

Ahnak is critical for cardiac Ca(V)1.2 calcium channel function and its beta-adrenergic regulation.

Defective L-type Ca2+ channel (I(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I(CaL) is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha1C(Ca(V)1.2)- and beta2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on I(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta2 subunit interaction and I(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta2 subunit decreased by approximately 50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on I(CaL) increasing the amplitude by approximately 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I(CaL). This genetic ahnak variant might cause individual differences in I(CaL) regulation upon physiological challenges or therapeutic interventions.

Constriction velocities of renal afferent and efferent arterioles of mice are not related to SMB expression.

BACKGROUND: Constriction of renal arterioles contributes significantly to the control of perfusion and glomerular filtration. Afferent but not efferent arterioles express smooth muscle myosin heavy chain B (SMB) (with a 5'-insert of seven amino acids). The aim of the present study was to investigate (1) the constriction characteristics of afferent and efferent arterioles under physiologic load and (2) whether expression of SMB may causally contribute to these constriction characteristics. METHODS: We compared constriction parameters [constriction amplitude, maximal rate of constriction velocity ("dc/dt(max)"), and time to half-maximal constriction (t(1/2)) of in vitro perfused renal afferent and efferent arterioles of wild-type (smb(+/+)] and homozygous SMB knockout [smb(-/-)] mice upon stimulation with angiotensin II (Ang II) (10(-8) mol/L) and potassium chloride (KCl) (100 mmol/L). SMB expression was investigated by double-labeling immunofluorescence. RESULTS: Contraction amplitude and dc/dt(max) of mouse afferent arterioles upon Ang II stimulation were significantly greater compared to efferent arterioles. However, constriction amplitudes, dc/dt(max), and t(1/2) of afferent as well as efferent arterioles upon Ang II stimulation were similar in smb(+/+) and smb(-/-) mice. Constriction amplitudes upon KCl stimulation of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. Furthermore, KCl-induced dc/dt(max) and t(1/2) of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. SMB expression could be detected in afferent but not efferent arterioles in smb(+/+) mice. No SMB expression in either arteriole could be observed in smb(-/-) mice. CONCLUSION: Our results suggest that the presence of different alternatively 5'-spliced smooth muscle-myosin heavy chain (SM-MHC) isoforms does not dominate the different contractile features of physiologically loaded renal afferent or efferent arterioles.

Reconstitution of ventricular myosin with atrial light chains 1 improves its functional properties.

Atrial light chain 1 (ALC-1) is expressed in embryonic and hypertrophied human ventricles but not in normal adult human ventricles. We investigated the effects of recombinant human atrial light chains (hALC-1) on the structure and enzymatic activity of synthetic filaments of ventricular myosin. The endogenous ventricular myosin light chain 1 (VLC-1) was partially replaced by recombinant hALC-1 yielding hALC-1 levels of 12%, 24% and 42%. This reconstitution of ventricular myosin with hALC-1 did not change the length of synthetic myosin filaments but led to more rounded myosin heads in comparison with those of control filaments. Actin-activated ATPase activity of myosin, a parameter of functional activity of molecular motor, amounted to 79.5 nmol P(i)/mg per min in control myosin filaments. Reconstitution with hALC-1 caused a profound increase of the actin-activated myosin ATPase activity in a dose dependent manner, for example, synthetic myosin filaments formed with 12%, 24% and 42% hALC-1 reconstituted myosin revealed the actin-activated ATPase activity increased by 18%, 26% and 36%, respectively, as compared to control. These results strongly suggest that in vivo expression of ALC-1 enhances ventricular myosin function, thereby contributing to cardiac compensation.

The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction.

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17 +/- 4 microg hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8 +/- 2.3 to 45.1 +/- 3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7 +/- 89.8 to 2,181 +/- 135.4 mmHg/s in the contraction rate, and 713 +/- 60.2 to 1,364 +/- 137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.