The pathological progression of hypertrophic cardiomyopathy (HCM) is sex 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, 10–12 month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca2+-sensitive tension development in demembranated cardiac trabeculae excised from 10–12 month female and male HCM mice. Whereas HCM did not impact Ca2+-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca2+ than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca2+-sensitive tension development was due to changes in Ca2+ handling and sarcomeric proteins, including expression of SR Ca2+ ATPase (2a) (SERCA2a), β-myosin heavy chain (β-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and β-MyHC protein whereas male HCM hearts showed a similar elevation of β-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.
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.
Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.
Contractile perturbations downstream of Ca2+ binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy “sensing” is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular “energy” sensing in the heart.
in myofilament Ca 2þ sensitivity was abolished by the expression of b-Tm. Furthermore, the McTnT 45-74 deletion-induced reduction in cooperativity of force production was more pronounced under a b-Tm background. Thus, our data shows that changes in the isoform expression of Tm modify T1dependent cardiac function, indicating that T1-Tm interactions exert a modulatory role in regulating cardiac thin filament activation. In the heart, stimulation of b-adrenergic pathway and subsequent activation of protein kinase A (PKA) is known to increase myocardial contractility. The increase in contractility is, in part, due to target phosphorylation of troponin I (TnI). In this study, we sought to identify novel target sites for PKA that could potentially contribute to this increase in contractility. To induce phosphorylation of TnI, cardiac and fast skeletal muscle from 3-4 month old Sprague Dawley rats was mechanically disrupted and demembranated followed by incubation with the catalytic subunit of PKA (50U PKA/ 3mg tissue , 0-30 min). To identify target specific phosphorylation on fast skeletal (fsTnI) or cardiac (cTnI) TnI, western blot analysis with phospho-specific antibodies was performed. PKA treatment increased phosphorylation of cTnI at ser22/23, as expected, but also at ser149. Similarly, PKA treatment increased phosphorylation of fsTnI at ser117, which is the equivalent to ser149 in cTnI. Accordingly, fsTnI demonstrated no observable phosphorylation at ser22/23. Adenosine-monophosphate activated kinase (AMPK) has been shown to target ser149 of cTnI. Therefore, to validate PKAdependent phosphorylation of cTnI at ser149, hearts were excised and perfused with AICAR, a known activator of AMPK. AICAR-perfused hearts demonstrated a time-dependent increase in phosphorylation of cTnI at ser149. These results demonstrate that PKA-dependent phosphorylation can target ser149 in cTnI and, equivalently, ser117 in fsTnI. The functional consequence of this target site phosphorylation and how it impacts contractility is currently under investigation.
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