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

Abstract

Restrictive cardiomyopathy (RCM) is a debilitating disease characterized by impaired ventricular filling, reduced ventricular volumes, and severe diastolic dysfunction. Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy and heightened risk of premature sudden cardiac death. These cardiomyopathies can result from mutations in the same gene that encodes for cardiac troponin I (cTnI). Acute genetic engineering of adult rat cardiac myocytes was used to ascertain whether primary physiologic outcomes could distinguish between RCM and HCM alleles at the cellular level. Co-transduction of cardiac myocytes with wild-type (WT) cTnI and RCM/HCM linked mutants in cTnI's inhibitory region (IR) demonstrated that WT cTnI preferentially incorporates into the sarcomere over IR mutants. The cTnI IR mutants exhibited minor effects in single, rodent, myocyte Ca 2+ -activated tension assays yet prolonged relaxation and Ca 2+ decay. RCM cTnI mutants in the helix-4/C-terminal region demonstrated a) hyper-sensitivity to Ca 2+ under loaded conditions, b) slower myocyte mechanical relaxation and Ca 2+ transient decay, c) frequency-dependent Ca 2+ -independent diastolic tone, d) heightened myofilament incorporation and e) irreversible cellular contractile defects with acute diltiazem administration. For species comparison, a subset of cTnI mutants were tested in isolated adult rabbit cardiac myocytes. Here, RCM and HCM mutant cTnIs exerted similar effects of slowed sarcomere length relaxation and Ca 2+ transient decay but did not show variable phenotypes by cTnI region. This study highlights cellular contractile defects by cardiomyopathy mutant cTnIs that are allele and species dependent. The species dependent results in particular raise important issues toward elucidating a unifying mechanistic pathway underling the inherited cardiomyopathies.

Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres

“…The results of this study are generally consistent with those from in vitro reconstituted protein and isolated experimental animal skinned myocardium studies, including the drastic increase in Ca 2ϩ sensitivity (9). Either a decrease (9) or, as in the present study, an increase (36) in maximum force development has been reported for the RCM-associated cTnI R145W mutation; most reports on the HCM-associated cTnI R145G mutation show a decrease in this parameter (10,11,35,37). Whether this reflects a genuine difference between the HCM-associated R145G mutation and the R145W RCM-associated mutation that we examined in the present study remains to be determined.…”

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

“…For example, the R92Q (56), R145G (57), R145W (58), ⌬160 (56), A172T (59), and R193H (9,60) mutations in cTnI all increase the myofilament Ca 2ϩ sensitivity and prolong SL relaxation times in isolated cardiomyocytes. Similarly, the older R21C mice have adapted by delaying the sarcomere relaxation time of myocytes, even in the absence of ␤-AR stimulation (Fig.…”

Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca2+-sensitive myofilaments

“…At the molecular level, HCM and RCM mutations have been shown to increase the Ca 2ϩ sensitivity of the myofilament (4,25). In vitro thin filament reconstitution experiments, with the only variable being the mutated protein, have shown that HCM and RCM mutations increase Ca 2ϩ sensitivity, with RCM mutations causing a larger increase than HCM mutations (2,4,34).…”

“…In vitro thin filament reconstitution experiments, with the only variable being the mutated protein, have shown that HCM and RCM mutations increase Ca 2ϩ sensitivity, with RCM mutations causing a larger increase than HCM mutations (2,4,34). From a molecular standpoint, these two diseases are a continuum of the same disease mechanism.…”

“…From a molecular standpoint, these two diseases are a continuum of the same disease mechanism. In general, RCM mutations result in a larger increase of myofilament Ca 2ϩ sensitivity causing a more severe phenotype (4,12,20). At the cellular level, myofilament mutations show altered sarcomere relaxation and/or heightened contractility and increased diastolic tone manifest by a pronounced precontractile state (5).…”