Thin Filament Disinhibition by Restrictive Cardiomyopathy Mutant R193H Troponin I Induces Ca ²⁺ -Independent Mechanical Tone and Acute Myocyte Remodeling

Abstract

Abstract-Inherited restrictive cardiomyopathy (RCM) is a debilitating disease characterized by a stiff heart with impaired ventricular relaxation. Mutations in cardiac troponin I (cTnI) were identified as causal for RCM. Acute genetic engineering of adult cardiac myocytes was used to identify primary structure/function effects of mutant cTnI. Studies focused on R193H cTnI owing to the poor prognosis of this allele. Compared with wild-type cTnI, R193H mutant cTnI more effectively incorporated into the sarcomere, where it exerted dose-dependent effects on basal and dynamic contractile function. Under loaded conditions, permeabilized myocyte Ca 2ϩ sensitivity of tension was increased, whereas the passive tension-extension relationship was not altered by R193H cTnI. Normal rod-shaped myocyte morphology acutely transitioned to a "short-squat" phenotype in concert with progressive stoichiometric incorporation of R193H in the absence of altered diastolic Ca 2ϩ . The specific myosin inhibitor blebbistatin fully blocked this transition. Heightened Ca 2ϩ buffering by the R193H myofilaments, and not alterations in Ca 2ϩ handling by the sarcoplasmic reticulum, slowed the decay rate of the Ca 2ϩ transient. Incomplete mechanical relaxation conferred by R193H was exacerbated at increasing pacing frequencies independent of elevated diastolic Ca 2ϩ . R193H cTnI-dependent mechanical tone caused acute remodeling to a quasicontracted state not elicited by other Ca 2ϩ -sensitizing proteins and is a direct correlate of the stiff heart characteristic of RCM in vivo. These results point toward targets downstream of Ca 2ϩ handling, notably thin filament regulation and actin-myosin interaction, in designing therapeutic strategies to redress the primary cell morphological and mechanical underpinnings of RCM. (Circ Res. 2007;100:1494-1502.) Key Words: cardiomyopathy Ⅲ troponin Ⅲ contraction I nherited cardiomyopathies represent a clinically diverse group of progressive heart muscle diseases that can be caused by mutations in specific sarcomeric genes. 1 Cardiomyopathies are classified into several distinct clinical subtypes based on a range of morphological and functional criteria. 1 The most malignant and least studied of the subtypes is restrictive cardiomyopathy (RCM). 2 The distinguishing clinical features of RCM patients include markedly impaired ventricular filling, pronounced diastolic dysfunction from an extremely stiff heart, 1,2 and the potential for rapid progression to overt heart failure. 2,3 Recently, mutations were identified in the gene encoding cardiac troponin I (cTnI), TNNI3, in human patients with autosomal dominant RCM. 3 RCM-linked mutant cTnIs result in amino acid substitutions in the most highly conserved regions of cTnI. 4 cTnI is known to function as a molecular switch within the sarcomere by regulating the Ca 2ϩ dependent cardiac muscle contraction. 4 During diastole, myocyte intracellular Ca 2ϩ is low and cTnI binds tightly to actin inhibiting strong actin-myosin interactions. Elevation of intracellular Ca...

Allele and species dependent contractile defects by restrictive and hypertrophic cardiomyopathy-linked troponin I mutants

“…These competition assays demonstrate that the IR mutant cTnIs are at a disadvantage relative to WT cTnI when incorporating into the sarcomere. This is in contrast to the recent report that RCM R193H cTnI significantly out-competed WT cTnI and preferentially incorporated into the intact sarcomere [22].…”

“…Control myocytes were able to fully relax and cycle Ca 2+ despite increasing frequency ( Figure 4A and B), but R146G myocytes yielded a frequency-dependent increase in diastolic Ca 2+ ( Figure 4B, right panel) and corresponding shortening of diastolic sarcomere length ( Figure 4B, left panel). Ca 2+ cycling occurs normally in the hyper Ca 2+ sensitized A172T myocyte ( Figure 4B), but with escalations in pacing frequency the A172T mutant myocytes developed a mechanical tone that was independent of [Ca 2+ ] during diastole ( Figure 4B), a result similar to that reported in RCM R193H myocytes [22].…”

“…To generate recombinant viral vectors, the mutant cTnIs were subcloned with and without C-terminal flag epitope into the pDC315 AdMax shuttle vector (Microbix Biosystems Inc.). Viral particle lysates were prepared for infection of a cell factory and plaque assays were used for determining the viral titer [22] …”

“…To compliment our previously published work on adult rat cardiac myocytes acutely engineered with RCM mutant R193H cTnI [22], adult rabbit myocytes transduced with R193H cTnI are also included in this comparative analysis. Similar to rat, targeted stoichiometric replacement of the native cTnI for recombinant mutant cTnI in rabbit cardiac myocytes was measured by Western blot ( Figure 6A) in which a time dependent increase in endogenous cTnI replacement was observed ( Figure 6A).…”

“…By contrast the primary physiologic consequence of most of the RCM mutant alleles remains poorly understood. While biochemical reconstitution models have shown that RCM cTnI alleles over sensitize the myofilament to Ca 2+ in both tension and ATPase assays [17; 20;21], a recent study using acute gene transfer to adult rodent cardiac myocytes with RCM R193H mutant allele demonstrated an increased efficiency of R193H mutant cTnI incorporation into the sarcomere over wild-type (WT) cTnI and a primary and unique evolution of a Ca 2+ independent diastolic tone and acute cellular remodeling in the absence of changes in Ca 2+ handling, a result not seen with other Ca 2+ sensitizing molecules [22]. Given these results, RCM mutants may alter the predicted dosage of mutant gene product and may affect myocyte physiology differently under intact Ca 2+ cycling conditions, particularly given the recent evidence that the interplay between Ca 2+ handling and cardiomyopathy mutant sarcomeres is critical to the organ level outcome [23].…”