Transmural variation in myosin heavy chain isoform expression modulates the timing of myocardial force generation in porcine left ventricle

Abstract

Recent studies have shown that the sequence and timing of mechanical activation of myocardium vary across the ventricular wall. However, the contributions of variable expression of myofilament protein isoforms in mediating the timing of myocardial activation in ventricular systole are not well understood. To assess the functional consequences of transmural differences in myofilament protein expression, we studied the dynamic mechanical properties of multicellular skinned preparations isolated from the sub-endocardial and sub-epicardial regions of the porcine ventricular midwall. Compared to endocardial fibres, epicardial fibres exhibited significantly faster rates of stretch activation and force redevelopment (k(tr)), although the amount of force produced at a given [Ca2+] was not significantly different. Consistent with these results, SDS-PAGE analysis revealed significantly elevated expression of alpha myosin heavy chain (MHC) isoform in epicardial fibres (13 +/- 1%) versus endocardial fibres (3 +/- 1%). Linear regression analysis revealed that the apparent rates of delayed force development and force decay following stretch correlated with MHC isoform expression (r2 = 0.80 and r2 = 0.73, respectively, P < 0.05). No differences in the relative abundance or phosphorylation status of other myofilament proteins were detected. These data show that transmural differences in MHC isoform expression contribute to regional differences in dynamic mechanical function of porcine left ventricles, which in turn modulate the timing of force generation across the ventricular wall and work production during systole.

Figure 2. Stretch activation responses of porcine skinned myocardium isolated from the endocardial and epicardial midwall

Force transients following a stretch of 1% of muscle length were recorded at maximal [Ca²⁺] activations in skinned fibres from the endocardium (En) and the epicardium (Ep). Once a steady-state isometric force was achieved at maximal Ca²⁺ activation, the muscle was stretched and then held at the longer length, as described in Methods. These representative transients are normalized to pre-stretch isometric force corresponding to the force base-line, which is arbitrarily set at zero. The recorded variables are labelled on the force record and described in the text.

Figure 1. Force–pCa relationships of endocardial and epicardial myocardium

Myocardium isolated from epicardial (•) and endocardial left ventricular midwall (^) displayed similar force–pCa relationships. Data points are means ±s.e.m. of 19 endocardial and 19 epicardial fibres.

Figure 3. Regional variation in myosin heavy chain (MHC) composition in porcine ventricular myocardium

Myosin heavy chain (MHC) isoform content was determined using 6% SDS-PAGE. The relative proportions of α and β MHC isoforms were determined by densitometric analysis gels following a silver-staining protocol. In this representative gel it can be seen that muscle fibres isolated from the endocardial midwall (lane 1 and lane 3) expressed less cardiac α MHC (3.5% and 2.2% of the total MHC content, respectively) than muscle fibres isolated from the epicardial midwall (lane 2 and lane 4) of the left ventricle (14.1% and 13.7% of the total MHC content, respectively).

Figure 4. Linear regression analysis of α myosin heavy chain (MHC) expression and stretch activation kinetics

The relationship between expression of α MHC (as percentage of total MHC content) and the apparent rates of stretch-induced delayed force development (k_df) (A) and force decay (k_rel) (B) was determined by linear regression analysis in 19 endocardial (^) and 19 epicardial (•) fibres. A significant positive correlation was found between increased α MHC expression and accelerated k_df and k_rel in the endocardial and epicardial midwall of porcine left ventricle which is described by the regression equations: k_df (s⁻¹) = 0.085 (%α MHC) + 1.901 (r²= 0.80, P < 0.05), and k_rel (s⁻¹) = 0.745 (%α MHC) + 17.463 (r²= 0.73, P < 0.05).

Figure 5. Determination of myofibrillar protein phosphorylation levels

A, representative SDS-PAGE (6 μg load) of the basal phosphorylation state of myofibrillar proteins in myocardium isolated from the endocardial (En) and epicardial (Ep) midwall. Preparation of porcine myocardial preparations loaded on the gel and analysis of phosphorylation status of proteins was performed as described in the Methods. SYPRO-Ruby stained gel for total proteins (left lanes) and Pro-Q Diamond-stained gel specific for phosphorylated proteins (right lanes). cMyBP-C, myosin binding protein-C; TnT, troponin T; TnI, troponin I; RLC, regulatory light chain. B, the slopes of protein and phosphoprotein determined from plots of area × mean raw optical density versus volume loaded for endocardial and epicardial myocardium. Different volumes of skinned endocardial and epicardial myocardial samples prepared from five porcine hearts were separated by SDS-PAGE and stained with SYPRO-Ruby for total proteins (top panel) and Pro-Q Diamond for phosphoproteins (bottom panel). The area and mean raw optical density (OD) of cMyBP-C and cTnI bands were determined and plotted against volume (μg) loaded. Regression lines were fitted to the data points and the resultant slope for proteins and phosphoproteins is shown in the top and bottom panel, respectively. Each data point represents the mean ±s.e.m.

Figure 5. Determination of myofibrillar protein phosphorylation levels

A, representative SDS-PAGE (6 μg load) of the basal phosphorylation state of myofibrillar proteins in myocardium isolated from the endocardial (En) and epicardial (Ep) midwall. Preparation of porcine myocardial preparations loaded on the gel and analysis of phosphorylation status of proteins was performed as described in the Methods. SYPRO-Ruby stained gel for total proteins (left lanes) and Pro-Q Diamond-stained gel specific for phosphorylated proteins (right lanes). cMyBP-C, myosin binding protein-C; TnT, troponin T; TnI, troponin I; RLC, regulatory light chain. B, the slopes of protein and phosphoprotein determined from plots of area × mean raw optical density versus volume loaded for endocardial and epicardial myocardium. Different volumes of skinned endocardial and epicardial myocardial samples prepared from five porcine hearts were separated by SDS-PAGE and stained with SYPRO-Ruby for total proteins (top panel) and Pro-Q Diamond for phosphoproteins (bottom panel). The area and mean raw optical density (OD) of cMyBP-C and cTnI bands were determined and plotted against volume (μg) loaded. Regression lines were fitted to the data points and the resultant slope for proteins and phosphoproteins is shown in the top and bottom panel, respectively. Each data point represents the mean ±s.e.m.

Figure 6. Myosin regulatory light chain (RLC) phosphorylation of porcine skinned myocardium

Two-dimensional SDS-PAGE/IEF gels were used to determine levels of RLC phosphorylation in endocardial (left panel) and epicardial (right panel) midwall skinned myocardium. In this representative gel two isoforms of ventricular light chain (VLC₂a and VLC₂b) and their corresponding phosphorylated isoforms (VLC₂a* and VLC₂b*) can be detected. It can be seen that VLC₂b and VLC₂a* co-migrate similarly and are not readily separated; however, densitometric scans of the RLC spots in gels from four porcine hearts indicated that phosphorylation levels were not significantly different between endocardial and epicardial myocardium with VLC₂a* and VLC₂b* representing 13.5 ± 3.1% and 16.8 ± 3.3% of the total VLC₂a and VLC₂b content in the endocardium and epicardium, respectively.