Daria Petzhold

Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β-adrenergic regulation

Defective L‐type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of β‐adrenergic receptors, PKA phosphorylates the α1C(CaV1.2)‐ and β2‐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 ICaL. Binding experiments with ahnak fragments (wild‐type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr‐ahnak critically affected both β2 subunit interaction and ICaL regulation. Binding affinity between ahnak‐C1 (aa 4646‐5288) and β2 subunit decreased by ≈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 ICaL increasing the amplitude by ≈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 ICaL by isoprenaline. Hence, we suggest the ahnak‐C1 domain serves as physiological brake on ICaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr‐ahnak fragments to increase ICaL. This genetic ahnak variant might cause individual differences in ICaL regulation upon physiological challenges or therapeutic interventions.—Haase, H., Alvarez, J., Petzhold, D., Doller, A., Behlke, J., Erdmann, J., Hetzer, R., Regitz‐Zagrosek, V., Vassort, G., Morano, I. Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β‐adrenergic regulation. FASEB J. 19, 1969–1977 (2005)

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.

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 (KD) for the hVLC‐1/1–15‐actin complex compared with the KD 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 μM) 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.–Haase, H., Dobbernack, G., Tünnemann, G., Karczewski, P., Cardoso, C., Petzhold, D., Schlegel, W.‐P., Lutter, S., Pierschalek, P., Behlke, J. Morano, I. Minigenes encoding N‐terminal domains of human cardiac myosin light chain‐1 improve heart function of transgenic rats. FASEB J. 20, 865–873 (2006)

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 β-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 β-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN−/−). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca2+ amplitude and maximal rate of Ca2+ rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) β-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 β-adrenergic stimulation of adult cardiomyocytes.

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.