Dilated cardiomyopathy (DCM) is a life-threatening heart disease significantly contributing to systolic heart failure and sudden cardiac death based on reduced cardiac contractility [1
]. Molecular and genetic studies have identified more than 30 different DCM disease genes, mainly coding for proteins of the sarcomere, the cardiac Z-disc and the cytoskeleton [5
To allow adaption of cardiac contractility on changing circulatory demands such as arterial blood pressure or volume preload, the autoregulatory cardiac stretch sensor system translates biomechanical strain of cardiomyocytes into activation of several signaling pathways regulating myocardial contractile force in vertebrates. Congruously, mutations of genes coding for proteins of the cardiac mechanical stress sensor are known to cause DCM in humans [6
]. However, the detailed genetic and molecular underpinnings of this complex autoregulatory mechanism are not fully understood yet, but of high clinical importance, since impaired adaption of cardiac contractility is considered to cause a sizeable proportion of DCM-related heart failure cases in humans [2
]. Genetic studies of cardiac stretch sensor components in zebrafish, mice and humans identified the integrin-linked kinase (ILK) as an essential regulator of cardiac contractility adaption on changing circulatory demands [7
In a forward genetic screen, we identified the zebrafish DCM mutant main squeeze
), carrying a mutation in the kinase domain of ILK (L308P), leading to reduced kinase activity and finally to a loss of cardiac stretch sensor function. Accordingly, homozygous msq
mutant embryos are characterized by severely reduced ventricular pump function as well as by decreased expression levels of stretch responsive genes such as the atrial natriuretic factor
) and vascular endothelial growth factor
]. Together with PINCH (particularly interesting Cys-His-rich protein) and β-parvin, ILK forms the functional ILK-PINCH-parvin (IPP) complex (Figure 1
], which is a crucial element of the cardiac stretch sensor [13
]. Similar to the ILK-deficient msq
mutant, ablation of β-parvin or PINCH in wild-type zebrafish leads to severely reduced cardiac contractility emphasizing that ILK as well as its interactors are essential regulators of ventricular pump function [15
In vertebrates, ILK is mainly expressed in heart and skeletal muscle, where it interacts through integrins with growth factor receptors and signaling molecules such as the protein kinase B (PKB) for signal transduction from the extracellular matrix to the cytoplasm [16
] (Figure 1
). In line with this, PKB phosphorylation as a downstream target of ILK is severely reduced in msq
zebrafish. Remarkably, overexpression of constitutive active PKB restores cardiac contractility of msq
], indicating that PKB phosphorylation is critical for regular heart function. However, efficient pharmacological approaches to enhance PKB phosphorylation and activation have not been established yet, but might be crucial to improve contractile performance in vivo.
In recent years, the zebrafish has emerged as a powerful tool for high-throughput in vivo screening of small chemical compounds allowing biomolecule evaluation with straightforward assessment of essential cardiac parameters such as cardiac development, myocardial contractility and heart rhythm [21
]. Using the zebrafish as drug screening platform, we aimed to identify chemical compounds rescuing heart failure in msq
mutant embryos via maintenance of PKB phosphorylation. Hence, by using our automated small compound screening platform, we identified two phosphatase inhibitors, okadaic acid and calyculin A, to significantly improve ventricular pump function by enhancing PKB phosphorylation in ILK-deficient msq
mutant zebrafish embryos.
Integrin-linked kinase is a key molecule of the mechanical stretch sensor in the vertebrate heart, regulating expression of stretch-responsive genes such as anf
and thereby allowing adaption of cardiac contractility to various hemodynamic demands. As shown in several genetic studies in animal models as well as in humans, mutations in genes encoding for proteins of the mechanical stretch sensor system lead to reduced ventricular FS and finally to dilated cardiomyopathy [2
Based on a mutation within the kinase domain of ILK homozygous mutant msq
embryos display a progressive reduction of myocardial contractility. On molecular level, homozygous msq
embryos are characterized by a reduced PKB phosphorylation and a decreased expression of the stretch responsive genes anf
], making msq
a suitable animal model for ILK-dependent DCM. Despite detailed characterization of the ILK-PKB signaling pathway, pharmacological approaches to restore ILK-PKB function are still missing.
With the aim to enhance ILK-PKB signaling pathway in msq, we studied the impact of 32 small compounds derived from a phosphatase inhibitor library on ventricular FS in this heart failure model. We hypothesized that the inhibition of dephosphorylation and consecutive inactivation of PKB by phosphatase inhibitors might lead to restored ILK-PKB signaling and finally to reconstituted cardiac contractility.
In our primary small compound screen using our recently established screening platform [21
], we evaluated 32 compounds and found three biomolecules, namely calyculin A, okadaic acid and cyclosporine A significantly improving ventricular FS. Interestingly, these compounds are known to act on the protein phosphatases PP1 and PP2A, which are essential regulators of the phosphorylation status in numerous key signaling pathways [30
]. Drug screening of other PP1/PP2A inhibitors such as the compounds B1 (cantharidic acid) and B2 (cantharidin) led to lethality of both wild-type and msq
mutant embryos. We conclude that our straightforward small compound screening approach allows to identify appropriate potential therapeutic biomolecules and to exclude substances with adverse effects from further in vivo evaluation.
As observed in our secondary drug screening, we found that among the three identified compounds calyculin A turned out to have the strongest effect on cardiac contractility and PKB phosphorylation. However, in contrast to okadaic acid, it has been shown that calyculin A inhibits not only PP1 and PP2A but also the myosin light chain (MLC) phosphatase, which is an important regulator of the contractile apparatus in the vertebrate heart [30
]. Myosin light chain phosphatase dephosphorylates the regulatory light chain of myosin II and initiates the relaxation process of muscle cells. Whether the inhibition of MLC phosphatase by calyculin A also contributes to restoration of ventricular FS in msq
mutants was not analyzed in our experimental setting. However, we conclude that, due to its pleiotropic effects in cardiomyocytes, calyculin A should be considered as a promising biomolecule with the potential to treat ILK-dependent DCM.
Cyclosporine A is an effective immunosuppressive drug that has been prescribed for decades for a vast number of patients, e.g., after organ transplantation to reduce graft-versus-host reactions.
In contrast to calyculin A and okadaic acid, cyclosporine A failed to significantly increase ventricular FS in msq
mutants despite recovered PKB phosphorylation. Hence, in our experimental setting cyclosporine A occurred only as an intermediate mediator of the ILK-PKB signaling pathway in comparison to calyculin A and okadaic acid. However, it is known that cyclosporine A has a beneficial myocardial effect by attenuating detrimental hypertrophy of the left ventricle in mice undergoing pressure overload [33
]. Hence, based on the protective effect in cardiomyopathy model organisms and the long-term experience with cyclosporine A in daily clinical routine, cyclosporine A is still an interesting target for future investigations in the context of cardiomyopathies.
In recent years, the zebrafish has emerged as a powerful and reliable model organism for the rapid and straightforward in vivo analysis of small molecule bioactivity for a broad range of cardiovascular diseases. Advances in the field like fully-automated high-throughput screenings will be of additional advantage, enabling testing of numerous biomolecules in an effective and time-saving manner [23
]. Although we show promising results of at least two compounds for the treatment of genetic ILK-dependent cardiomyopathy in a vertebrate model, additional studies in alternative model systems are needed to elucidate the transferability of our results to mammals and humans.
In this context, human-induced-pluripotent-stem-cell-derived cardiomyocytes cells (hiPSC-CM) have been successfully established in recent years for cardiovascular disease modeling as well as drug screening. In contrast to animal models, hiPSC-CM are biologically identical to their human donors, facilitating significantly the transferability of novel genetic and molecular findings. However, hiPSC-CM differ in several important aspects from adult human cardiomyocytes, especially in terms of maturation, gene expression or ion channel function, reducing their field of application [35
]. In contrast, large mammalian animal models for cardiomyopathies, such as dogs, render pathomechanistical findings that are easy to transfer to humans based on the high interspecies homogeneities, but large-scale drug screening in a cost and time saving manner is not applicable in this type of model organism. Thus, we conclude that the different disease models, including cell-based approaches as well as vertebrate and mammalian animal models, with their particular strengths and weaknesses, should be seen as complementary in long-term drug discovery rather than exclusionary.
Initial drug screening with 32 compounds was partly performed with an arbitrarily predefined biomolecule concentration of 10 µM. This one-concentration-fits-all approach facilitates evaluating numerous compounds in a short time. However, biological impact of compounds that were lethal in our animal model might be overestimated and biological impact of compounds with no obvious effect on ventricular FS might be underestimated in our experimental setting due to inappropriate compound concentration.