Length and PKA Dependence of Force Generation and Loaded Shortening in Porcine Cardiac Myocytes

Abstract

In healthy hearts, ventricular ejection is determined by three myofibrillar properties; force, force development rate, and rate of loaded shortening (i.e., power). The sarcomere length and PKA dependence of these mechanical properties were measured in porcine cardiac myocytes. Permeabilized myocytes were prepared from left ventricular free walls and myocyte preparations were calcium activated to yield ~50% maximal force after which isometric force was measured at varied sarcomere lengths. Porcine myocyte preparations exhibited two populations of length-tension relationships, one being shallower than the other. Moreover, myocytes with shallow length-tension relationships displayed steeper relationships following PKA. Sarcomere length-K(tr) relationships also were measured and K(tr) remained nearly constant over ~2.30 μm to ~1.90 μm and then increased at lengths below 1.90 μm. Loaded-shortening and peak-normalized power output was similar at ~2.30 μm and ~1.90 μm even during activations with the same [Ca(2+)], implicating a myofibrillar mechanism that sustains myocyte power at lower preloads. PKA increased myocyte power and yielded greater shortening-induced cooperative deactivation in myocytes, which likely provides a myofibrillar mechanism to assist ventricular relaxation. Overall, the bimodal distribution of myocyte length-tension relationships and the PKA-mediated changes in myocyte length-tension and power are likely important modulators of Frank-Starling relationships in mammalian hearts.

Figure 1

Sarcomere length-tension relationships in porcine skinned ventricular cardiac myocyte preparations. (a) Muscle cell preparations were mounted between a force transducer and motor, calcium activated to yield ~50% maximal force, then isometric force was measured over a range of sarcomere lengths monitored using an IonOptix SarLen system. (b) Histogram showing the slopes of length-tension relationships obtained in porcine cardiac myocytes.

Figure 2

(a) Pig cardiac myocyte sarcomere length-tension relationships before and after PKA treatment. PKA-induced phosphorylation markedly steepened the length-tension relationship. (b) An autoradiogram showing radiolabeled phosphate incorporation into pig cardiac myofibrillar proteins (MyBP-C and cTnI) upon PKA treatment. Without PKA treatment, there was no radiolabelled ATP incorporation (data not shown).

Figure 3

Sarcomere length dependence of the rate constant of force redevelopment (k _tr⁡). (a) Slow time-based recordings of sarcomere length and force obtained using an IonOptix SarLen system during a slack restretch maneuver during submaximal Ca²⁺ activation. (b) Sarcomere length-dependence of k _tr⁡ for rat slow-twitch skeletal muscle fibers, rat cardiac myocytes, and pig cardiac myocytes. Although pig cardiac myocyte k _tr⁡ was much slower at all sarcomere lengths compared to rat cardiac myocytes, both pig and rat cardiac cell preparations showed that k _tr⁡ increased at short sarcomere lengths despite reductions in force implicating that sarcomere length overrides the Ca²⁺ activation dependence of k _tr⁡.

Figure 4

Normalized force-velocity and power-load curves from a pig left ventricular myocyte preparation at long and short sarcomere length obtained during half-maximal Ca²⁺ activations. Pig cardiac myocyte preparations exhibited little sarcomere length dependence of loaded shortening and power output. Inset shows bar plot (mean ± SD) of peak normalized power output at long (~2.30 μm) and short (~1.90 μm) sarcomere length (n = 8 myocyte preparations).

Figure 5

(a) Silver-stained gel showing the myosin heavy chain isoforms contained in a rat cardiac myocyte preparation compared to a pig cardiac myocyte preparation. (b) Representative length and force traces during a lightly loaded force clamp in a rat cardiac myocyte preparation (red) and a pig cardiac myocyte preparation (black) during a submaximal Ca²⁺ activation. (c) Length traces exhibited considerably greater curvature (greater k _shortening) in rat myocytes compared to pig myocyte preparations at all relative loads. Inset in C shows an expanded plot of k _shortening versus relative load, which clarifies slope constants below 0.30. Interestingly, PKA-mediated phosphorylation increased the curvature (k _shortening) of length traces towards those of rat myocyte preparations. In addition, pig cardiac myocyte preparations that exhibited steep length-tension relationships (L-T R) also had more curved length traces. This is consistent with PKA-mediated phosphorylation of myofilaments yielding greater responsiveness to changes in sarcomere length, in this case exhibited by greater shortening-induced cooperative deactivation.

Figure 6

Normalized force-velocity and power-load curves from a pig left ventricular myocyte preparation before and after PKA treatment during half-maximal Ca²⁺ activations. Pig cardiac myocyte preparations exhibited more power after PKA treatment. Inset shows bar plot (mean ± SD) of peak normalized power output before and after PKA (n = 10 myocyte preparations).

Figure 7

(a) Representative Frank-Starling relationship from one animal at baseline (Base) and after treatment with dobutamine (DBT). (b) Comprehensive group data from all animals illustrating a significant leftward shift in the Frank-Starling relationship (mixed model, treatment main effect adjusted for EDV covariance, P < 0.05). There was no significant interaction or change in slope of the Frank-Starling relationship between treatments (see table inset in (b)), therefore, parallelism was assumed. The y-intercept and marginal mean difference were both significantly increased following the dobutamine treatment (*P < 0.05; table inset (b)). The dobutamine challenge resulted in a ~15% increase in stroke volume (SV) for a given end diastolic volume (EDV) in    vivo. This increase in ventricular function was similar in magnitude to that observed in our myocyte preparations (~10%), illustrating the coherence of our whole heart and cardiac myocyte functional data.

Figure 8

Representative amplitude-normalized calcium transients of Pig ((a)–(c)) and Mouse (d) left-ventricular myocytes (0.5 Hz, field stimulus denoted by arrow). Calcium transients from Pig exhibited multiple waveforms, including normal recovery from the transient peak (a, 2 of 14 cells), recovery with a marked shoulder ((b), 8 of 14 cells), and recovery with a secondary increase in calcium (c, 4 of 14 cells). (d) Mouse transients consistently exhibited a rapid transient recovery (n = 40). (e) Overlay of transients shown in (b) and (d) illustrating the distinct transient kinetics between pig (gray) and mouse (black) myocytes.