Aaron C. Hinken

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to β1-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following β1-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in β1-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca2+ tension and Ca2+-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca2+-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca2+ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to β1-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.

Desensitization of Myofilaments to Ca2+ as a Therapeutic Target for Hypertrophic Cardiomyopathy with Mutations in Thin Filament Proteins

Background—Hypertrophic cardiomyopathy (HCM) is a common genetic disorder caused mainly by mutations in sarcomeric proteins and is characterized by maladaptive myocardial hypertrophy, diastolic heart failure, increased myofilament Ca2+ sensitivity, and high susceptibility to sudden death. We tested the following hypothesis: correction of the increased myofilament sensitivity can delay or prevent the development of the HCM phenotype. Methods and Results—We used an HCM mouse model with an E180G mutation in &agr;-tropomyosin (Tm180) that demonstrates increased myofilament Ca2+ sensitivity, severe hypertrophy, and diastolic dysfunction. To test our hypothesis, we reduced myofilament Ca2+ sensitivity in Tm180 mice by generating a double transgenic mouse line. We crossed Tm180 mice with mice expressing a pseudophosphorylated cardiac troponin I (S23D and S24D; TnI-PP). TnI-PP mice demonstrated a reduced myofilament Ca2+ sensitivity compared with wild-type mice. The development of pathological hypertrophy did not occur in mice expressing both Tm180 and TnI-PP. Left ventricle performance was improved in double transgenic compared with their Tm180 littermates, which express wild-type cardiac troponin I. Hearts of double transgenic mice demonstrated no changes in expression of phospholamban and sarcoplasmic reticulum Ca2+ ATPase, increased levels of phospholamban and troponin T phosphorylation, and reduced phosphorylation of TnI compared with Tm180 mice. Moreover, expression of TnI-PP in Tm180 hearts inhibited modifications in the activity of extracellular signal-regulated kinase and zinc finger-containing transcription factor GATA in Tm180 hearts. Conclusions—Our data strongly indicate that reduction of myofilament sensitivity to Ca2+ and associated correction of abnormal relaxation can delay or prevent development of HCM and should be considered as a therapeutic target for HCM.

Phosphorylation of Cardiac Troponin I at Protein Kinase C Site Threonine 144 Depresses Cooperative Activation of Thin Filaments

There is evidence for PKC-dependent multisite phosphorylation of cardiac troponin I (cTnI) at Ser-23 and Ser-24 (also PKA sites) in the cardiac-specific N-terminal extension and at Thr-144, a unique residue in the inhibitory region. The functional effect of these phosphorylations in combination is of interest in view of data indicating intramolecular interaction between the N-terminal extension and the inhibitory region of cTnI. To determine the role of PKC-dependent phosphorylation of cTnI on sarcomeric function, we measured contractile regulation at multiple levels of complexity. Ca2+ binding to thin filaments reconstituted with either cTnI(wild-type) or pseudo-phosphorylated cTnI(S23D/S24D), cTnI(T144E), and cTnI(S23D/S24D/T144E) was determined. Compared with controls regulated by cTnI(wild-type), thin filaments with cTnI(S23D/S24D) and cTnI(S23D/S24D/T144E) exhibited decreased Ca2+ sensitivity. In contrast, there was no significant difference between Ca2+ binding to thin filaments with cTnI(wild-type) and with cTnI(T144E). Studies of the pCa-force relations in skinned papillary fibers regulated by these forms of cTnI yielded similar results. However, in both the Ca2+ binding measurements and the skinned fiber tension measurements, the presence of cTnI(S23D/S24D/T144E) induced a much lower Hill coefficient than either wild type, S23D/S24D, or T144E. These data highlight the importance of thin filament-based cooperative mechanisms in cardiac regulation, with implications for mechanisms of control of function in normal and pathological hearts.

Functional effects of a tropomyosin mutation linked to FHC contribute to maladaptation during acidosis

Familial hypertrophic cardiomyopathy (FHC) is a leading cause of sudden cardiac death among young athletes but the functional effects of the myofilament mutations during FHC-associated ischemia and acidosis, due in part to increased extravascular compressive forces and microvascular dysfunction, are not well characterized. We tested the hypothesis that the FHC-linked tropomyosin (Tm) mutation Tm-E180G alters the contractile response to acidosis via increased myofilament Ca(2+) sensitivity. Intact papillary muscles from transgenic (TG) mice expressing Tm-E180G and exposed to acidic conditions (pH 6.9) exhibited a significantly smaller decrease in normalized isometric tension compared to non-transgenic (NTG) preparations. Times to peak tension and to 90% of twitch force relaxation in TG papillary muscles were significantly prolonged. Intact single ventricular TG myocytes demonstrated significantly less inhibition of unloaded shortening during moderate acidosis (pH 7.1) than NTG myocytes. The peak Ca(2+) transients were not different for TG or NTG at any pH tested. The time constant of re-lengthening was slower in TG myocytes, but not the rate of Ca(2+) decline. TG detergent-extracted fibers demonstrated increased Ca(2+) sensitivity of force and maximal tension compared to NTG at both normal and acidic pH (pH 6.5). Tm phosphorylation was not different between TG and NTG muscles at either pH. Our data indicate that acidic pH diminished developed force in hearts of TG mice less than in NTG due to their inherently increased myofilament Ca(2+) sensitivity, thus potentially contributing to altered energy demands and increased propensity for contractile dysfunction.

New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I.

Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca(2+)-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca(2+)-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca(2+) and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.

The Fast Skeletal Troponin Activator, CK-1909178 Reduces Muscle Fatigue in a Model of Peripheral Artery Disease in Situ

CK-1909178 is a member of a class of fast skeletal troponin activators that sensitize skinned skeletal muscle fibers to calcium. In rat muscle preparations in vitro and in situ, CK-1909178 increased sub-tetanic force without altering maximum force. Given that a major cause of muscle fatigue during repeated muscle contraction is reduced myoplasmic Ca2+ due to impaired sarcoplasmic reticulum Ca2+ release, we tested whether increased calcium sensitivity with CK-1909178 would slow the development of fatigue. Rat flexor digitorum brevis muscle was pretreated in vitro with CK-1909178 and stimulated every 3 seconds at a frequency sufficient to achieve 50% of maximum force for 6 min at 30°C. CK-1909178 diminished the extent of fatigue as compared to control (terminal force 29.5±8% vs. 12.7±4%, p<0.001). We next tested whether CK-1909178 treatment would slow the development of muscle fatigue using rat extensor digitorum longus muscle in situ, where the muscle was stimulated via the peroneal nerve. To accelerate the development of muscle fatigue, vascular insufficiency was produced by femoral artery ligation (FAL). Muscle fatigue with FAL and sham ligation in the presence and absence of CK-1909178 was assessed. CK-1909178 was administered as a 5mg/kg intravenous bolus before assessment of fatigue at a frequency adjusted to achieve the same force at 30Hz prior to dosing. FAL resulted in significantly reduced terminal tension as compared to sham (33±4% vs. 77±5%, p<0.01). CK-1909178 administration significantly attenuated FAL-induced fatigue at 10 minutes (61±7% vs. 33±4%, p<0.01). In summary, CK-1909178 increased sub-maximal muscle force development and reduced the extent of fatigue in the presence of limited blood flow in situ. We believe that this mechanism may improve muscle fatigue in diseases where blood flow to muscles is compromised such as intermittent claudication.