Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. Methods: We generated the knock-in mice ( Mylk3 ^+/fs and Mylk 3 ^fs/fs ) with a familial dilated cardiomyopathy–associated MYLK3 frameshift mutation ( MYLK3 ^+/fs ) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell–derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). Results: Both mice ( Mylk3 ^+/fs and Mylk 3 ^fs/fs ) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose–dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_ MYLK3 vector. Human induced pluripotent stem cell–derived cardiomyocytes with MYLK3 _+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_ MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V _max for ventricular myosin regulatory light chain phosphorylation without affecting the K _m . LEUO-1154 treatment of human induced pluripotent stem cell–derived cardiomyocytes with MYLK3 _+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3 / PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. Conclusions: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Mutations in sarcomere genes can cause both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). However, the complex genotype-phenotype relationships in pathophysiology of cardiomyopathies by gene or mutation location are not fully understood. In addition, it is still unclear how mutations within same molecule result in different clinical phenotypes such as HCM and DCM. To clarify how the initial functional insult caused by a subtle change in one protein component of the sarcomere with a given mutation is critical for the development of proper effective treatments for cardiomyopathies. Fortunately, recent technological advances and the development of direct sarcomere modulators have provided a more detailed understanding of the molecular mechanisms that govern the effects of specific mutations. The direct inhibition of sarcomere contractility may be able to suppress the development and progression of HCM with hypercontractile mutations and improve clinical parameters in patients with HCM. On the other hand, direct activation of sarcomere contractility appears to exert unexpected beneficial effects such as reverse remodeling and lower heart rate without increasing adverse cardiovascular events in patients with systolic heart failure due to DCM. Direct sarcomere modulators that can positively influence the natural history of cardiomyopathies represent promising treatment options.

Comparative CO2 flux measurements by eddy covariance technique using open- and closed-path gas analysers over the equatorial Pacific Ocean