Kerry S. McDonald

Hypertrophic Cardiomyopathy in Cardiac Myosin Binding Protein-C Knockout Mice

Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (+/−) and homozygous (−/−) for the knockout allele. Wild-type (+/+), cMyBP-C+/−, and cMyBP-C−/− mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C+/− mice were indistinguishable from wild-type littermates, cMyBP-C−/− mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C−/− mice. Ca2+ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C−/− but not cMyBP-C+/− mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function.

Small Amounts of α-Myosin Heavy Chain Isoform Expression Significantly Increase Power Output of Rat Cardiac Myocyte Fragments

Myocardial performance is likely affected by the relative expression of the two myosin heavy chain (MyHC) isoforms, namely &agr;-MyHC and &bgr;-MyHC. The relative expression of each isoform is regulated developmentally and in pathophysiological states. Many pathophysiological states are associated with small shifts in the relative expression of each MyHC isoform, yet the functional consequence of these shifts remains unclear. The purpose of this study was to determine the functional effect of a small shift in the relative expression of &agr;-MyHC. To this end, power output was measured in rat cardiac myocyte fragments that expressed ≈12% &agr;-MyHC and in myocyte fragments that expressed ≈0% &agr;-MyHC, as determined in the same cells by SDS-PAGE analysis after mechanical experiments. Myocyte fragments expressing ≈12% &agr;-MyHC developed ≈52% greater peak normalized power output than myocyte fragments expressing ≈0% &agr;-MyHC. These results indicate that small amounts of &agr;-MyHC expression significantly augment myocyte power output.

Loaded Shortening, Power Output, and Rate of Force Redevelopment Are Increased With Knockout of Cardiac Myosin Binding Protein-C

Abstract— Myosin binding protein-C (MyBP-C) is localized to the thick filaments of striated muscle where it appears to have both structural and regulatory functions. Importantly, mutations in the cardiac MyBP-C gene are associated with familial hypertrophic cardiomyopathy. The purpose of this study was to examine the role that MyBP-C plays in regulating force, power output, and force development rates in cardiac myocytes. Skinned cardiac myocytes from wild-type (WT) and MyBP-C knockout (MyBP-C−/−) mice were attached between a force transducer and position motor. Force, loaded shortening velocities, and rates of force redevelopment were measured during both maximal and half-maximal Ca2+ activations. Isometric force was not different between the two groups with force being 17.0±7.2 and 20.5±3.1 kN/m2 in wild-type and MyBP-C−/− myocytes, respectively. Peak normalized power output was significantly increased by 26% in MyBP-C−/− myocytes (0.15±0.01 versus 0.19±0.03 P/Po · ML/sec) during maximal Ca2+ activations. Interestingly, peak power output in MyBP-C−/− myocytes was increased to an even greater extent (46%, 0.09±0.03 versus 0.14±0.02 P/Po · ML/sec) during half-maximal Ca2+ activations. There was also an effect on the rate constant of force redevelopment (ktr) during half-maximal Ca2+ activations, with ktr being significantly greater in MyBP-C−/− myocytes (WT=5.8±0.9 s−1 versus MyBP-C−/−=7.7±1.7 s−1). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.

Ca2+ dependence of loaded shortening in rat skinned cardiac myocytes and skeletal muscle fibres

1 This study examined the effects of activator Ca2+ on loaded shortening and power output in skinned rat cardiac myocyte preparations, and fast‐ and slow‐twitch skeletal muscle fibres at 12 °C. 2 Shortening velocities were slowed at nearly all relative loads when Ca2+ activation levels were reduced to ≈70 % maximal isometric force (P4.5) in cardiac myocyte preparations, as well as in fast‐twitch and slow‐twitch skeletal muscle fibres. 3 Peak absolute power outputs declined significantly as Ca2+ activation levels were progressively reduced from maximal to 30 %P4.5 in all three striated muscle types, with the greatest change in fast‐twitch fibres. In cardiac myocyte preparations, even peak relative power output progressively fell when Ca2+ activation levels were lowered to ≈70, 50 and 30 %P4.5. Peak relative power output also progressively fell in fast‐twitch fibres as Ca2+ activation levels were lowered from maximal down to 50 %P4.5. However, in slow‐twitch fibres, peak relative power output decreased only at 70 %P4.5 and then remained unchanged with further reductions in Ca2+ activation levels. The greater Ca2+ dependence of peak relative power output in cardiac myocytes and fast‐twitch fibres may arise from a shared mechanism such as cooperative inactivation of the thin filament, which is likely to be slowest in less cooperative slow‐twitch fibres. 4 During submaximal Ca2+ activations, the time course of shortening became markedly curvilinear during isotonic shortening in all three muscle types. The progressive slowdown in shortening velocity during isotonic contractions was greatest in fast‐twitch fibres, consistent with the higher degree of cooperativity of Ca2+ activation in fast‐twitch fibres. Additionally, fast‐twitch and slow‐twitch fibre stiffness decreased in concert with the curvature of length traces during loaded shortening. These results are consistent with the idea that cooperative inactivation of the thin filament occurs during loaded shortening and such a mechanism may contribute to the progressive slowing and overall Ca2+ dependence of loaded shortening velocity.

Force‐velocity and power‐load curves in rat skinned cardiac myocytes

1 This study utilized a skinned myocyte preparation with low end compliance to examine force‐velocity and power‐load curves at 12 °C in myocytes from rat hearts. 2 In maximally activated myocyte preparations, shortening velocities appeared to remain constant during load clamps in which shortening took place over a sarcomere length range of ≈2.30‐2.00 μm. These results suggest that previously reported curvilinear length traces during load clamps of multicellular preparations were due in part to extracellular viscoelastic structures that give rise to restoring forces during myocardial shortening. 3 During submaximal Ca2+ activations, the velocity of shortening at low loads slowed and the time course of shortening became curvilinear, i.e. velocity progressively slowed as shortening continued. This result implies that cross‐bridge cycling kinetics are slower at low levels of activation and that an internal load arises during shortening of submaximally activated myocytes, perhaps due to slowly detaching cross‐bridges. 4 Reduced levels of activator Ca2+ also reduced maximal power output and increased the relative load at which power output was optimal. For a given absolute load, the shift has the effect of maintaining power output near the optimum level despite reductions in cross‐bridge number and force generating capability at lower levels of Ca2+.

Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain

The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either α‐myosin heavy chain (α‐MyHC) or β‐MyHC at 12 ± 1°C. When SL was decreased from 2.3 μm to 2.0 μm submaximal isometric force decreased ∼40% in both α‐MyHC and β‐MyHC myocytes while peak absolute power output decreased 55% in α‐MyHC myocytes and 70% in β‐MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in β‐MyHC as in α‐MyHC myocytes (41%versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca2+] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in α‐MyHC myocytes, whereas in β‐MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both α‐MyHC and β‐MyHC myocytes. The rate constant of force development (ktr) was also measured and was not different between long and short SL at the same [Ca2+] in α‐MyHC myocytes but was greater at short SL in β‐MyHC myocytes. At short SL with matched force by either dextran or [Ca2+], ktr was greater than at long SL in both α‐MyHC and β‐MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and β‐MyHC myocytes exhibit a greater sarcomere length dependence of power output.

Heart failure with preserved ejection fraction: chronic low-intensity interval exercise training preserves myocardial O2 balance and diastolic function

We have previously reported chronic low-intensity interval exercise training attenuates fibrosis, impaired cardiac mitochondrial function, and coronary vascular dysfunction in miniature swine with left ventricular (LV) hypertrophy (Emter CA, Baines CP. Am J Physiol Heart Circ Physiol 299: H1348-H1356, 2010; Emter CA, et al. Am J Physiol Heart Circ Physiol 301: H1687-H1694, 2011). The purpose of this study was to test two hypotheses: 1) chronic low-intensity interval training preserves normal myocardial oxygen supply/demand balance; and 2) training-dependent attenuation of LV fibrotic remodeling improves diastolic function in aortic-banded sedentary, exercise-trained (HF-TR), and control sedentary male Yucatan miniature swine displaying symptoms of heart failure with preserved ejection fraction. Pressure-volume loops, coronary blood flow, and two-dimensional speckle tracking ultrasound were utilized in vivo under conditions of increasing peripheral mean arterial pressure and β-adrenergic stimulation 6 mo postsurgery to evaluate cardiac function. Normal diastolic function in HF-TR animals was characterized by prevention of increased time constant of isovolumic relaxation, normal LV untwisting rate, and enhanced apical circumferential and radial strain rate. Reduced fibrosis, normal matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-4 mRNA expression, and increased collagen III isoform mRNA levels (P < 0.05) accompanied improved diastolic function following chronic training. Exercise-dependent improvements in coronary blood flow for a given myocardial oxygen consumption (P < 0.05) and cardiac efficiency (stroke work to myocardial oxygen consumption, P < 0.05) were associated with preserved contractile reserve. LV hypertrophy in HF-TR animals was associated with increased activation of Akt and preservation of activated JNK/SAPK. In conclusion, chronic low-intensity interval exercise training attenuates diastolic impairment by promoting compliant extracellular matrix fibrotic components and preserving extracellular matrix regulatory mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype in a large animal model resembling heart failure with preserved ejection fraction.

Length dependence of force generation exhibit similarities between rat cardiac myocytes and skeletal muscle fibres

According to the Frank–Starling relationship, increased ventricular volume increases cardiac output, which helps match cardiac output to peripheral circulatory demand. The cellular basis for this relationship is in large part the myofilament length–tension relationship. Length–tension relationships in maximally calcium activated preparations are relatively shallow and similar between cardiac myocytes and skeletal muscle fibres. During twitch activations length–tension relationships become steeper in both cardiac and skeletal muscle; however, it remains unclear whether length dependence of tension differs between striated muscle cell types during submaximal activations. The purpose of this study was to compare sarcomere length–tension relationships and the sarcomere length dependence of force development between rat skinned left ventricular cardiac myocytes and fast‐twitch and slow‐twitch skeletal muscle fibres. Muscle cell preparations were calcium activated to yield ∼50% maximal force, after which isometric force and rate constants (ktr) of force development were measured over a range of sarcomere lengths. Myofilament length–tension relationships were considerably steeper in fast‐twitch fibres compared to slow‐twitch fibres. Interestingly, cardiac myocyte preparations exhibited two populations of length–tension relationships, one steeper than fast‐twitch fibres and the other similar to slow‐twitch fibres. Moreover, myocytes with shallow length–tension relationships were converted to steeper length–tension relationships by protein kinase A (PKA)‐induced myofilament phosphorylation. Sarcomere length–ktr relationships were distinct between all three cell types and exhibited patterns markedly different from Ca2+ activation‐dependent ktr relationships. Overall, these findings indicate cardiac myocytes exhibit varied length–tension relationships and sarcomere length appears a dominant modulator of force development rates. Importantly, cardiac myocyte length–tension relationships appear able to switch between slow‐twitch‐like and fast‐twitch‐like by PKA‐mediated myofibrillar phosphorylation, which implicates a novel means for controlling Frank–Starling relationships.

TEAD-1 Overexpression in the Mouse Heart Promotes an Age-dependent Heart Dysfunction

TEA domain transcription factor-1 (TEAD-1) is essential for proper heart development and is implicated in cardiac specific gene expression and the hypertrophic response of primary cardiomyocytes to hormonal and mechanical stimuli, and its activity increases in the pressure-overloaded hypertrophied rat heart. To investigate whether TEAD-1 is an in vivo modulator of cardiac specific gene expression and hypertrophy, we developed transgenic mice expressing hemagglutinin-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that a sustained increase in TEAD-1 protein leads to an age-dependent dysfunction. Magnetic resonance imaging revealed decreases in cardiac output, stroke volume, ejection fraction, and fractional shortening. Isolated TEAD-1 hearts revealed decreased left ventricular power output that correlated with increased βMyHC protein. Histological analysis showed altered alignment of cardiomyocytes, septal wall thickening, and fibrosis, although electrocardiography displayed a left axis shift of mean electrical axis. Transcripts representing most members of the fetal heart gene program remained elevated from fetal to adult life. Western blot analyses revealed decreases in p-phospholamban, SERCA2a, p-CX43, p-GSK-3α/β, nuclear β-catenin, GATA4, NFATc3/c4, and increased NCX1, nuclear DYKR1A, and Purα/β protein. TEAD-1 mice did not display cardiac hypertrophy. TEAD-1 mice do not tolerate stress as they die over a 4-day period after surgical induction of pressure overload. These data provide the first in vivo evidence that increased TEAD-1 can induce characteristics of cardiac remodeling associated with cardiomyopathy and heart failure.

Sarcomere length dependence of power output is increased after PKA treatment in rat cardiac myocytes.

The Frank-Starling relationship of the heart yields increased stroke volume with greater end-diastolic volume, and this relationship is steeper after beta-adrenergic stimulation. The underlying basis for the Frank-Starling mechanism involves length-dependent changes in both Ca(2+) sensitivity of myofibrillar force and power output. In this study, we tested the hypothesis that PKA-induced phosphorylation of myofibrillar proteins would increase the length dependence of myofibrillar power output, which would provide a myofibrillar basis to, in part, explain the steeper Frank-Starling relations after beta-adrenergic stimulation. For these experiments, adult rat left ventricles were mechanically disrupted, permeabilized cardiac myocyte preparations were attached between a force transducer and position motor, and the length dependence of loaded shortening and power output were measured before and after treatment with PKA. PKA increased the phosphorylation of myosin binding protein C and cardiac troponin I, as assessed by autoradiography. In terms of myocyte mechanics, PKA decreased the Ca(2+) sensitivity of force and increased loaded shortening and power output at all relative loads when the myocyte preparations were at long sarcomere length ( approximately 2.30 mum). PKA had less of an effect on loaded shortening and power output at short sarcomere length ( approximately 2.0 mum). These changes resulted in a greater length dependence of myocyte power output after PKA treatment; peak normalized power output increased approximately 20% with length before PKA and approximately 40% after PKA. These results suggest that PKA-induced phosphorylation of myofibrillar proteins explains, in part, the steeper ventricular function curves (i.e., Frank-Starling relationship) after beta-adrenergic stimulation of the left ventricle.