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Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal‐regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.

Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal–regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (If) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ releases. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, where heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ specific manner to maintain cardiac energy homeostasis and determine cardiac physiological adaptation to exercise, by modulating intrinsic sinoatrial cell behaviour.

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerisation in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescencebased sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnI(R145G) on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI(WT) or hcTnI(R145G). Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI(R146G) mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mm BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca(2)(+). Maximum force development (F(max)) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI(R145G) in vitro. Ca(2)(+) sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k(ACT) is reduced at maximal [Ca(2)(+)] but not at low [Ca(2)(+)] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k(REL). We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signalling-dependent hyperphagia, obesity and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that longterm AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3β treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

Pathogenesis of most myopathies including inherited hypertrophic (HCM) and dilated (DCM) cardiomyopathies is based on modification of structural state of contractile proteins induced by point mutations, such as mutations in alpha-tropomyosin (TM). To understand the mechanism of abnormal function of contractile system of muscle fiber due to Glu180Gly, Asp175 or Glu40Lys, Glu54Lys mutations in alpha-TM associated with HCM or DCM, we specifically labeled alpha-TM by fluorescence probe 5-IAF after Cys-190 and examined the position and mobility of the IAF-TM in the ATP hydrolysis cycle using polarized fluorescence technique. Analysis of the data suggested that the point mutations in alpha-TM associated with hypertrophic or dilated cardiomyopathy caused abnormal changes in the affinity ofTM to actin and in the position of this protein on the thin filaments in the ATPase cycle. Mutations in alpha-TM associated with HCM caused a shift of TM strands to the center of the thin filament and increased a range of tropomyosin motion and affinity of this protein to actin in the ATPase cycle. In contrast, mutations in alpha-TM associated with DCM shifted the protein to the periphery of the thin filament, reduced the amplitude of the TM movement and its affinity for actin. It is proposed that anomalous behavior of TM on the thin filaments in ATPase cycle may provoke the dysfunction of the cardiac muscle in patients with HCM and DCM.