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Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse
Jennifer Davis1, Soichiro Yasuda, Nathan J Palpant
1Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Abstract:
Restrictive cardiomyopathy (RCM) has been linked to mutations in the thin filament regulatory protein cardiac troponin I (cTnI). As the pathogenesis of RCM from genotype to clinical phenotype is not fully understood, transgenic (Tg) mice were generated with cardiac specific expression of an RCM-linked missense mutation (R193H) in cTnI. R193H Tg mouse hearts with 15% stoichiometric replacement had smaller hearts and significantly elevated end diastolic pressures (EDP) in vivo. Using a unique carbon microfiber-based assay, membrane intact R193H adult cardiac myocytes generated higher passive tensions across a range of physiologic sarcomere lengths resulting in significant Ca(2+) independent cellular diastolic tone that was manifest in vivo as elevated organ-level EDP. Sarcomere relaxation and Ca(2+) decay was uncoupled in isolated R193H Tg adult myocytes due to the increase in myofilament Ca(2+) sensitivity of tension, decreased passive compliance of the sarcomere, and adaptive in vivo changes in which phospholamban (PLN) content was decreased. Further evidence of Ca(2+) and mechanical uncoupling in R193H Tg myocytes was demonstrated by the biphasic response of relaxation to increased pacing frequency versus the negative staircase seen with Ca(2+) decay. In comparison, non-transgenic myocyte relaxation closely paralleled the accelerated Ca(2+) decay. Ca(2+) transient amplitude was also significantly blunted in R193H Tg myocytes despite normal mechanical shortening resulting in myocyte hypercontractility when compared to non-transgenics. These results identify for the first time that a single point mutation in cTnI, R193H, directly causes elevated EDP due to a myocyte intrinsic loss of compliance independent of Ca(2+) cycling or altered cardiac morphology. The compound influence of impaired relaxation and elevated EDP represents a clinically severe form of diastolic dysfunction similar to the hemodynamic state documented in RCM patients.
Insights
A cardiac troponin I (cTnI) mutation (R193H) causes restrictive cardiomyopathy by increasing diastolic tone and impairing relaxation in heart cells. This leads to elevated end-diastolic pressure, mimicking clinical RCM symptoms.
Area of Science:
- Cardiology
- Molecular Biology
- Genetics
Background:
- Restrictive cardiomyopathy (RCM) is linked to mutations in cardiac troponin I (cTnI).
- The genotype-to-phenotype relationship in RCM remains unclear.
- Understanding the molecular mechanisms of RCM is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the functional consequences of an RCM-linked cTnI mutation (R193H) in a cardiac-specific transgenic mouse model.
- To elucidate the cellular and organ-level mechanisms by which the R193H mutation contributes to diastolic dysfunction.
- To determine if the R193H mutation directly causes elevated end-diastolic pressure (EDP) independent of altered cardiac morphology.
Main Methods:
- Generation of cardiac-specific transgenic (Tg) mice expressing the R193H missense mutation in cTnI.
- In vivo assessment of cardiac function, including end-diastolic pressure (EDP).
- In vitro analysis of isolated adult cardiac myocytes using a carbon microfiber-based assay to measure passive tension, Ca(2+) sensitivity, and relaxation dynamics.
Main Results:
- R193H Tg mouse hearts exhibited smaller size and significantly elevated EDP.
- Isolated R193H Tg myocytes showed increased passive tension and Ca(2+)-independent diastolic tone.
- Impaired myocyte relaxation and Ca(2+) decay uncoupling were observed, alongside blunted Ca(2+) transient amplitude and hypercontractility.
Conclusions:
- The R193H cTnI mutation directly induces elevated EDP through a myocyte-intrinsic loss of compliance, independent of Ca(2+) cycling or cardiac morphology.
- Impaired relaxation and elevated EDP in R193H Tg myocytes recapitulate the severe diastolic dysfunction seen in RCM patients.
- This study identifies a specific molecular mechanism linking cTnI mutations to RCM pathogenesis.
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