LKB1/Mo25/STRAD uniquely impacts sarcomeric contractile function and posttranslational modification.

The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca(2+) sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca(2+) sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.

Temporal and morphological impact of pressure overload in transgenic FHC mice.

Although familial hypertrophic cardiomyopathy (FHC) is characterized as cardiac disease in the absence of overt stressors, disease penetrance, and pathological progression largely depend on modifying factors. Accordingly, pressure overload by transverse aortic constriction (TAC) was induced in 2-month-old, male mice with and without a FHC (R403Q) mutation in α-myosin heavy chain. A significantly greater number of FHC mice (n = 8) than wild-type (WT) mice (n = 5) died during the 9-week study period. TAC induced a significant increase in cardiac mass whether measured at 2 or 9 weeks post-TAC in both WT and FHC mice, albeit to a different extent. However, the temporal and morphological trajectory of ventricular remodeling was impacted by the FHC transgene. Both WT and FHC hearts responded to TAC with an early (2 weeks post-TAC) and significant augmentation of the relative wall thickness (RWT) indicative of concentric hypertrophy. By 9 weeks post-TAC, RWT decreased in WT hearts (eccentric hypertrophy) but remained elevated in FHC hearts. WT hearts following TAC demonstrated enhanced cardiac function as measured by the end-systolic pressure-volume relationship, pre-load recruitable stroke work (PRSW), and myocardial relaxation indicative of compensatory hypertrophy. Similarly, TAC induced differential histological and cellular remodeling; TAC reduced expression of the sarcoplasmic reticulum Ca(2+)-ATPase (2a) (SERCA2a; 2 and 9 weeks) and phospholamban (PLN; 2 weeks) but increased PLN phosphorylation (2 weeks) and ß-myosin heavy chain (ß-MyHC; 9 weeks) in WT hearts. FHC-TAC hearts showed increased ß-MyHC (2 and 9 weeks) and a late (9 weeks) decrease in PLN expression concomitant with a significant increase in PLN phosphorylation. We conclude that FHC hearts respond to TAC induced pressure overload with increased premature death, severe concentric hypertrophy, and a differential ability to undergo morphological, functional, or cellular remodeling compared to WT hearts.

Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice.

The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10-12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca(2+)-sensitive tension development in demembranated cardiac trabeculae excised from 10-12month female and male HCM mice. Whereas HCM did not impact Ca(2+)-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca(2+) than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca(2+)-sensitive tension development was due to changes in Ca(2+) handling and sarcomeric proteins, including expression of SR Ca(2+) ATPase (2a) (SERCA2a), ß-myosin heavy chain (ß-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and ß-MyHC protein whereas male HCM hearts showed a similar elevation of ß-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS-PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.

Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25.

BACKGROUND: Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress. METHODOLOGY/PRINCIPAL FINDINGS: Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist. CONCLUSIONS/SIGNIFICANCE: Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.