The effects of slow skeletal troponin I expression in the murine myocardium are influenced by development‐related shifts in myosin heavy chain isoform

Abstract

Key points• Slow skeletal troponin I (ssTnI) transgenic (TG) mice were treated with propylthiouracil (PTU) to induce a shift in myosin heavy chain (MHC) from the α-to β-MHC isoform, to understand how concomitant expression of these proteins affects cardiac muscle function.• Following PTU treatment, β-MHC expression increased to ∼80%, relative to α-MHC while TG ssTnI expression persisted at a level of ∼34% of total TnI.• ssTnI sped XB recruitment dynamics, and this increase was enhanced ∼3.8-fold in the presence of β-MHC when compared to ssTnI effects against α-MHC.• The ssTnI effect to increase myofilament Ca 2+ sensitivity was blunted in the presence of β-MHC.• Our results provide new evidence for significant TnI-MHC interactions in their effects on cardiac function, which has major implications for coupling between concerted expression of contractile regulatory isoforms and the thick and thin filament-mediated tuning of cardiac contractile function.Abstract Troponin I (TnI) and myosin heavy chain (MHC) are two contractile regulatory proteins that undergo major shifts in isoform expression as cardiac myocytes mature from embryonic to adult stages. To date, many studies have investigated individual effects of embryonic vs. cardiac isoforms of either TnI or MHC on cardiac muscle function and contractile dynamics. Thus, we sought to determine whether concomitant expression of the embryonic isoforms of both TnI and MHC had functional effects that were not previously observed. Adult transgenic (TG) mice that express the embryonic isoform of TnI, slow skeletal TnI (ssTnI), were treated with propylthiouracil (PTU) to revert MHC expression from adult (α-MHC) to embryonic (β-MHC) isoforms. Cardiac muscle fibres from these mice contained ∼80% β-MHC and ∼34% ssTnI of total MHC or TnI, respectively, allowing us to test the functional effects of ssTnI in the presence of β-MHC. Detergent-skinned cardiac muscle fibre bundles were used to study how the interplay between MHC and TnI modulate muscle length-mediated effect on crossbridge (XB) recruitment dynamics, Ca 2+ -activated tension, and ATPase activity. One major finding was that the model-predicted XB recruitment rate (b) was enhanced significantly by ssTnI, and this speeding effect of ssTnI on XB recruitment rate was much greater (3.8-fold) when β-MHC was present. Another major finding was that the previously documented ssTnI-mediated increase in myofilament Ca 2+ sensitivity (pCa 50 ) was blunted when β-MHC was present. ssTnI expression increased pCa 50 by 0.33 in α-MHC fibres, whereas ssTnI increased pCa 50 by only 0.05 in β-MHC fibres. Our study provides new evidence for significant interplay between MHC and TnI isoforms that is essential for tuning cardiac contractile function. Thus, MHC-TnI interplay may provide a developmentally dependent mechanism to enhance XB recruitment dynamics at a time when Ca 2+ -handling mechanisms are underdeveloped, and to prevent excessive ssTnI-dependent inotropy (increased Ca 2+ sensitivity) in the embryonic myocardium.

Altered Right Ventricular Mechanical Properties Are Afterload Dependent in a Rodent Model of Bronchopulmonary Dysplasia

“…In normoxic trabeculae, the rate of force redevelopment ( k tr ) varied with the level of activating [Ca 2+ ] free (or force), increasing as [Ca 2+ ] free (or force) was elevated from sub-maximal to maximal levels (Figures 4 , 5 ). These Ca 2+ - and force-dependent changes in k tr in normoxic trabeculae are consistent with previous results from rat (Wolff et al, 1995 ; Palmer and Kentish, 1998 ; Olsson et al, 2004 ; Patel et al, 2012 ), mice (Edes et al, 2007 ; Colson et al, 2012 ; Ford and Chandra, 2012 ), porcine (Edes et al, 2007 ) and human (Edes et al, 2007 ) myocardium. Both, 35-day normoxic and hyperoxic trabeculae also exhibited similar Ca 2+ - and force-dependent changes in k tr , suggesting no remaining significant effects of hyperoxia on these relationships.…”

“…While these results are consistent with the idea that higher density of thick and thin filaments allows 21-day hyperoxic trabeculae to generate more force, the finding of similar amount of maximum Ca 2+ activated forces in 35-day hyperoxic and normoxic trabeculae is inconsistent with this mechanism. Previous studies reported that myocardium expressing ssTnI generates a maximum Ca 2+ activated force similar to myocardium expressing cTnI (Fentzke et al, 1999 ; Arteaga et al, 2000 ; Konhilas et al, 2003 ; Ford and Chandra, 2012 ) whereas expression of aMLC1 results in a higher maximum Ca 2+ activated force than expression of vMLC1 (Morano et al, 1998 ). Interestingly, our findings of increased maximum Ca 2+ activated force and aMLC1 in 21-day old hyperoxia exposed rats was associated with PH, which is similar to a report in a porcine model of PH (Morano et al, 1998 ).…”

“…Thus, the possibility of the depressed cross-bridge cycling kinetics in 21-day hyperoxic trabeculae may be exclusively or in part due to a decrease in expression of α-MHC and concomitant increase in expression of β-MHC (Figure 6B ). Previous studies have reported that expression of ssTnI has no significant effects on the rate of force redevelopment in skinned preparations (Ford and Chandra, 2012 ), whereas expression of aMLC1 increases contraction and relaxation time in whole heart (Morano et al, 1996 , 1998 ; Fewell et al, 1998 ; Abdelaziz et al, 2004 ). Since there is expression of aMLC1 and ssTnI in 21-day hyperoxic myocardium but not in normoxic myocardium, both aMLC1and ssTnI appears to be responsible for slower contraction kinetics in hyperoxic myocardium at this age.…”

“…At sub-maximal Ca 2+ , 21-day hyperoxic trabeculae generated more force than age matched normoxic trabeculae and as a result, the force-pCa relationships established in hyperoxic trabeculae were left-shifted by ~0.23 pCa units compared to those in normoxic trabeculae (Figure 3A ), which corresponds to similar changes found in monocrotaline-induced PH (Kogler et al, 2003 ). In cardiac muscle, expression of either ssTnI (Fentzke et al, 1999 ; Arteaga et al, 2000 ; Konhilas et al, 2003 ; Ford and Chandra, 2012 ) or aMLC1 (Morano et al, 1997 ; Diffee and Nagle, 2003 ; Diffee, 2004 ) are known to shift force-pCa relationships to the left. Since 21-day hyperoxic trabeculae expressed both ssTnI and aMLC1, it is difficult to say with certainty that the increased Ca 2+ sensitivity of force in 21-day hyperoxic trabeculae was due to the presence of ssTnI or aMLC1, or both.…”

Interplay Between the Effects of Dilated Cardiomyopathy Mutation (R206L) and the Protein Kinase C Phosphomimic (T204E) of Rat Cardiac Troponin T Are Differently Modulated by α‐ and β‐Myosin Heavy Chain Isoforms

“…However, our data showed that both k tr and b remained unaltered, suggesting a discrepancy between our observations on g and the XB recruitment rates. However, this apparent discrepancy may be resolved by considering that f plays a much greater influence than g on k tr ; therefore, a slight decrease in g need not result in a decrease in k tr . Two other important observations to note from our estimates of XB recruitment dynamics are: (1) T204E decreased both k tr (Figure C) and b (Figure D) in α‐MHC+T204E fibers, but not in β‐MHC+T204E fibers, demonstrating that β‐MHC over‐rides the influence of T204E on XB recruitment dynamics; and (2) in α‐MHC+T204E+R206L fibers, both k tr (Figure C) and b (Figure D) were unaltered, which suggests that R206L negates the attenuating effect of T204E on XB turnover rates in α‐MHC fibers.…”