Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse model of restrictive cardiomyopathy

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 Ca²⁺ independent cellular diastolic tone that was manifest in vivo as elevated organ-level EDP. Sarcomere relaxation and Ca²⁺ decay was uncoupled in isolated R193H Tg adult myocytes due to the increase in myofilament Ca²⁺ sensitivity of tension, decreased passive compliance of the sarcomere, and adaptive in vivo changes in which phospholamban (PLN) content was decreased. Further evidence of Ca²⁺ 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 Ca²⁺ decay. In comparison, non-transgenic myocyte relaxation closely paralleled the accelerated Ca²⁺ decay. Ca²⁺ 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 Ca²⁺ 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.