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

Abstract

Background:The R21C mutation in cardiac troponin I (cTnI) prevents PKA-mediated phosphorylation of serines 23 and 24 of cTnI in vivo. Results: Myofilament function is uncoupled from the intracellular [Ca 2ϩ ] and delays muscle relaxation. Conclusion: Long term ablation of cTnI phosphorylation leads to hypertrophy, diastolic dysfunction, and dysautonomia in mice. Significance: Restoration of phosphorylated cTnI may prevent hypertrophic cardiomyopathy and diastolic dysfunction.The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca 2؉ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca 2؉ -transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca 2؉ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca 2؉ content. This abnormal Ca 2؉ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na ؉ -Ca 2؉ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.Inherited as an autosomal dominant disease, familial hypertrophic cardiomyopathy (HCM) 2 is the most common genetic disorder of the heart (1). This clinical syndrome is characterized by an increase in left ventricular mass, diastolic dysfunction, and dysautonomia and carries a high incidence of sudden cardiac death (2). In many HCM cases, cardiac contractile dysfunction is attributed to inherited sarcomeric gene mutations, which can clinically present with variable penetrance, even within the same family pedigree (3,4). Within the context of the sarcomere, it is well established that mutations in cardiac troponin (cTn) alter t...

TNNI1, TNNI2 and TNNI3: Evolution, regulation, and protein structure–function relationships

“…cTnI-R21C knock-in mice showed diastolic dysfunction with delayed Ca 2+ re-sequestration (Dweck et al, 2014), a phenotype that supports the role of the N-terminal extension of cTnI in the regulation of diastolic function of the heart.…”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…Dweck et al . found that the isolated cardiac myocytes from R21C transgenic mice (with ages older than 12 months) significantly delayed Ca 2+ transient decay and relaxation [158]. The phenotype of cTnI R21C mutation supports the regulatory role of cTnI N-terminus in diastolic function of the heart.…”

“…Importantly, both mutants blunted the ability of PKA to reduce K C-I and pCa 50 and speed relaxation of myofibrils [125]. It is worth mentioning that cTnI R21C disrupts the PKA phosphorylation on S23/S24 of cTnI [30, 158, 185], and thus results in a “blunted” β-adrenergic stimulation response, which may be the actual physiologic/pathogenic mechanism of cTnI R21C . Therefore, to further understand whether it is the cTnI R21C per se that is altering function or just the inability to get S23/S24 phosphorylated, we introduced the bis -phosphomimic substitutions S23D/S24D into cTnI R21C (cTnI R21C/S23D/S24D ) to mimic effect of PKA phosphorylation.…”

Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation

“…19–33 Recently, we also reported the blunted β-adrenergic response for both cTnI HCM-associated mutations. 34,35 Both mutations significantly left-shifted (increased) Ca 2+ binding affinity to cTn ( K Ca ) and the affinity of cTnC for cTnI ( K C–I ).…”