Functional Effects of the H1-Helix of Rat Cardiac Troponin T on Crossbridge Detachment Rate is Differently Modulated by α- and β-Myosin Heavy Chain Isoforms

Chandra

2016

JAHA

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BackgroundWe hypothesized that the functional effects of R206L—a rat analog of the dilated cardiomyopathy (DCM) mutation R205L in human cardiac troponin T (TnT)—were differently modulated by myosin heavy chain (MHC) isoforms and T204E, a protein kinase C (PKC) phosphomimic of TnT. Our hypothesis was based on two observations: (1) α‐ and β‐MHC differentially influence the functional effects of TnT; and (2) PKC isoforms capable of phosphorylating TnT are upregulated in failing human hearts.Methods and ResultsWe generated 4 recombinant TnT variants: wild type; R206L; T204E; and R206L+T204E. Functional effects of the TnT variants were tested in cardiac muscle fibers (minimum 14 per group) from normal (α‐MHC) and propylthiouracil‐treated rats (β‐MHC) using steady‐state and dynamic contractile measurements. Notably, in α‐MHC fibers, Ca2+‐activated maximal tension was attenuated by R206L (≈32%), T204E (≈63%), and R206L+T204E (≈64%). In β‐MHC fibers, maximal tension was unaffected by R206L, but was attenuated by T204E (≈33%) and R206L+T204E (≈40%). Thus, β‐MHC differentially counteracted the attenuating effects of the TnT variants on tension. However, in β‐MHC fibers, R206L+T204E attenuated tension to a greater extent when compared to T204E alone. In β‐MHC fibers, R206L+T204E attenuated the magnitude of the length‐mediated recruitment of new cross‐bridges (≈28%), suggesting that the Frank‐Starling mechanism was impaired.ConclusionsOur findings are the first (to our knowledge) to demonstrate that the functional effects of a DCM‐linked TnT mutation are not only modulated by MHC isoforms, but also by the pathology‐associated post‐translational modifications of TnT.

mentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Interplay Between the Effects of Dilated Cardiomyopathy Mutation (R206L) and the Protein Kinase C Phosphomimic (T204E) of Rat Cardiac Troponin T Are Differently Modulated by α‐ and β‐Myosin Heavy Chain Isoforms

Chandra

2016

JAHA

Self Cite

Interplay between the effects of a Protein Kinase C phosphomimic (T204E) and a dilated cardiomyopathy mutation (K211Δ or R206W) in rat cardiac troponin T blunts the magnitude of muscle length-mediated crossbridge recruitment against the β-myosin heavy chain background

J Muscle Res Cell Motil

Gollapudi

Chandra

2016

Failing hearts of dilated cardiomyopathy (DCM)-patients reveal systolic dysfunction and upregulation of several Protein Kinase C (PKC) isoforms. Recently, we demonstrated that the functional effects of T204E, a PKC phosphomimic of cardiac troponin T (TnT), were differently modulated by α- and β-myosin heavy chain (MHC) isoforms. Therefore, we hypothesized that the interplay between the effects of T204E and a DCM-linked mutation (K211Δ or R206W) in TnT would modulate contractile parameters linked-to systolic function in an MHC-dependent manner. To test our hypothesis, five TnT variants (wildtype, K211Δ, K211Δ + T204E, R206W, and R206W + T204E) were generated and individually reconstituted into demembranated cardiac muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rats. Steady-state and mechano-dynamic measurements were performed on reconstituted fibers. Myofilament Ca(2+) sensitivity (pCa50) was decreased by both K211Δ and R206W to a greater extent in α-MHC fibers (~0.15 pCa units) than in β-MHC fibers (~0.06 pCa units). However, T204E exacerbated the attenuating influence of both mutants on pCa50 only in β-MHC fibers. Moreover, the magnitude of muscle length (ML)-mediated crossbridge (XB) recruitment was decreased by K211Δ + T204E (~47 %), R206W (~34 %), and R206W + T204E (~36 %) only in β-MHC fibers. In relevance to human hearts, which predominantly express β-MHC, our data suggest that the interplay between the effects of DCM mutations, PKC phosphomimic in TnT, and β-MHC lead to systolic dysfunction by attenuating pCa50 and the magnitude of ML-mediated XB recruitment.

Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and β-myosin heavy chain with sinusoidal analysis

Kawai

Karam

J Muscle Res Cell Motil

et al. 2016

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express β-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing ~99% α-MHC were treated with propylthiouracil to result in 100% β-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and β-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5 mM MgATP, 1 mM Mg, 8 mM Pi, 200 mM ionic strength, pH 7.00 at 25 °C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100 Hz (corresponding time domain: 1.6-1200 ms), and effects of Ca, Pi, and ATP were studied. The results show that Ca sensitivity was slightly less (10-15%) in β-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca activation) demonstrated that, the apparent rate constants were 2-4× faster in α-MHC containing fibers. The ATP study demonstrated that, in β-MHC containing fibers, K (ATP association constant) was greater (1.7×), k and k (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in β-MHC containing fibers, k (rate constant of the force-generation step) and k were smaller (0.75× and 0.25×, respectively), resulting in greater K (3×). There were no differences in active tension, rigor stiffness, or K (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K (Pi association constant).