Myofibrillar regulatory mechanisms of stretch activation in mammalian striated muscle

Dr. Kerry McDonald, Thesis Supervisor.Field of study: Medical pharmacology and physiology."May 2017."Stretch activation is described as a delayed increase in force after an imposed stretch. This process is essential in the flight muscles of many insects and is also observed, to some degree, in mammalian striated muscles. The mechanistic basis for stretch activation remains uncertain, although it appears to involve cooperative activation of the thin filaments (12, 80). The purpose of this study was to address myofibrillar regulatory mechanisms of stretch activation in mammalian striated muscle. For these studies, permeabilized rat slow-twitch and fast-twitch skeletal muscle fibers were mounted between a force transducer and motor, and a slack-re-stretch maneuver was performed over a range of Ca[superscript 2+] activation levels. Following slack-re-stretch there was a stretch activation process that often resulted in a transient force overshoot (P[subscript TO]), which was quantified relative to steady-state isometric force. P[subscript TO] was highly dependent upon Ca[superscript 2+] activation level, and the relative magnitude of P[subscript TO] was greater in slow-twitch fibers than fast-twitch fibers. In both slow-twitch and fast-twitch fibers, force redevelopment involved a fast, Ca[superscript 2+] activation dependent process (k1) and a slower, less activation dependent process (k2). Interestingly, the two processes converged at low levels of Ca[superscript 2+] activation in both fiber types. P[subscript TO] also contained a relaxation phase, which progressively slowed as Ca[superscript 2+] activation levels increased and was more Ca[superscript 2+] activation dependent in slow-twitch fibers. These results suggest that stretch activation may not be solely regulated by the extent of apparent cooperative activation of force due to a higher relative level of stretch activation in the less cooperative slow-twitch skeletal muscle fiber. Next, we investigated an additional potential molecular mechanism by regulating stretch activation in mammalian striated muscle. Along these lines, our lab has previously observed that PKA-induced phosphorylation of cMyBP-C and cTnI elicited a significant increase in transient force overshoot following slack-re-stretch maneuver in permeabilized cardiac myocytes (29). Interestingly, in slow-twitch skeletal muscle fibers MyBP-C but not ssTnI is phosphorylated by PKA (28). We, thus, took advantage of this variation in substrates phosphorylated by PKA to investigate the effects of PKA-induced phosphorylation of MyBP-C on stretch activation in slow-twitch skeletal muscle fibers. Following PKA treatment of skinned slow-twitch skeletal muscle fibers, the magnitude of P[subscript TO] more than doubled, but this only occurred at low levels of Ca[superscript 2+] activation (i.e., [approximately]25% maximal Ca[superscript 2+] activated force). Also, force redevelopment rates were significantly increased over the entire range of Ca[superscript 2+] activation levels following PKA treatment. In a similar manner, force decay rates showed a tendency of being faster following PKA treatment, however, were only statistically significantly faster at 50% Ca[superscript 2+] activation. Overall, these results are consistent with a model whereby stretch transiently increases the number of cross-bridges made available for force generation and PKA phosphorylation of MyBP-C enhances these stretch activation processes.Includes bibliographical references (pages 48-59)

Regulation of Myofilament Contractile Function in Human Donor and Failing Hearts

Heart failure (HF) often includes changes in myocardial contractile function. This study addressed the myofibrillar basis for contractile dysfunction in failing human myocardium. Regulation of contractile properties was measured in cardiac myocyte preparations isolated from frozen, left ventricular mid-wall biopsies of donor (n = 7) and failing human hearts (n = 8). Permeabilized cardiac myocyte preparations were attached between a force transducer and a position motor, and both the Ca2+ dependence and sarcomere length (SL) dependence of force, rate of force, loaded shortening, and power output were measured at 15 ± 1°C. The myocyte preparation size was similar between groups (donor: length 148 ± 10 μm, width 21 ± 2 μm, n = 13; HF: length 131 ± 9 μm, width 23 ± 1 μm, n = 16). The maximal Ca2+-activated isometric force was also similar between groups (donor: 47 ± 4 kN⋅m–2; HF: 44 ± 5 kN⋅m–2), which implicates that previously reported force declines in multi-cellular preparations reflect, at least in part, tissue remodeling. Maximal force development rates were also similar between groups (donor: ktr = 0.60 ± 0.05 s–1; HF: ktr = 0.55 ± 0.04 s–1), and both groups exhibited similar Ca2+ activation dependence of ktr values. Human cardiac myocyte preparations exhibited a Ca2+ activation dependence of loaded shortening and power output. The peak power output normalized to isometric force (PNPO) decreased by ∼12% from maximal Ca2+ to half-maximal Ca2+ activations in both groups. Interestingly, the SL dependence of PNPO was diminished in failing myocyte preparations. During sub-maximal Ca2+ activation, a reduction in SL from ∼2.25 to ∼1.95 μm caused a ∼26% decline in PNPO in donor myocytes but only an ∼11% change in failing myocytes. These results suggest that altered length-dependent regulation of myofilament function impairs ventricular performance in failing human hearts