Laurin M. Hanft

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.

Length dependence of striated muscle force generation is controlled by phosphorylation of cTnI at serines 23/24

•  An important mechanism in beat‐to‐beat optimization of heart performance is matching ventricular output with end‐diastolic volume, which is known as the Frank–Starling Relationship. •  The cellular basis for this regulation involves myofilament length–tension relationships. •  We previously showed two populations of length–tension relationships in mammalian left ventricular cardiac myocytes, one steep like fast‐twitch skeletal muscle fibres and the other shallow like slow‐twitch skeletal muscle fibres, and cardiac myocytes with shallow length–tension relationships shift to steep relationships by protein kinase A‐induced myofilament phosphorylation. •  The current study investigated the molecular and amino acid residue mechanisms that control length–tension relationships. •  The single muscle cell experiments demonstrated that cardiac troponin I phosphorylation at serines 23/24 control length–tension relationships in striated muscle. •  This study provides: (i) a mechanism to explain a length dependence of force generation in striated muscle; and (ii) an important target to potentially treat heart disease associated with compromised Frank–Starling relationships.

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

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 [Ca2+], 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.

Titin-mediated control of cardiac myofibrillar function

According to the Frank-Starling relationship, ventricular pressure or stroke volume increases with end-diastolic volume. This is regulated, in large part, by the sarcomere length (SL) dependent changes in cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both cardiac myofibrillar force and absolute power fall at shorter SL. However, when Ca(2+) activated force levels are matched between short and long SL (by increasing the activator [Ca(2+)]), short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that, at short SL, titin becomes less taut, which increases the flexibility of the cross-bridges, a process that may be mediated by titins interactions with thick filament proteins. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL-dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted that, in N2BA titin containing cardiac myocytes, power-load curves would not be shifted upward at short SL compared to long SL (when force is matched). Consistent with this, peak normalized power was actually less at short SL versus long SL (at matched force) in N2BA-containing myocytes (N2BA titin: ΔPNPO (Short SL peak power minus long SL peak power)=-0.057±0.049 (n=5) versus N2B titin: ΔPNPO=+0.012±0.012 (n=5). These findings support a model whereby SL per se controls mechanical properties of cross-bridges and this process is mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.

Dantrolene suppresses spontaneous Ca2+ release without altering excitation-contraction coupling in cardiomyocytes of aged mice.

Cardiac dysfunction in the aged heart reflects abnormalities in cardiomyocyte Ca(2+) homeostasis including altered Ca(2+) cycling through the sarcoplasmic reticulum (SR). The ryanodine receptor antagonist dantrolene exerts antiarrhythmic effects by preventing spontaneous diastolic Ca(2+) release from the SR. We tested the hypothesis that dantrolene prevents spontaneous Ca(2+) release without altering excitation-contraction coupling in aged myocardium. Left ventricular cardiomyocytes isolated from young (3 to 4 mo) and aged (24-26 mo) C57BL/6 mice were loaded with the Ca(2+) indicator fluo-4. Amplitudes of action potential-induced Ca(2+) transients at 1-Hz pacing were similar between young and aged mice, yet cell shortening was impaired in aged mice. Isoproterenol (1 μM) increased Ca(2+) transient amplitude and cell shortening to identical levels in young and aged; dantrolene (1 μM) had no effect on Ca(2+) transients or cell shortening during pacing. Under Ca(2+) overload conditions induced with 10 mM extracellular Ca(2+) concentration, spontaneous Ca(2+) waves were of diminished amplitude and associated with lower SR Ca(2+) content in aged versus young mice. Despite no effect in young mice, dantrolene increased SR Ca(2+) content and Ca(2+) wave amplitude in aged mice. In the presence of isoproterenol following rest from 1-Hz pacing, Ca(2+) spark frequency was elevated in aged mice, yet the time to spontaneous Ca(2+) wave was similar between young and aged mice; dantrolene decreased Ca(2+) spark frequency and prolonged the time to Ca(2+) wave onset in aged mice with no effect in young mice. Thus dantrolene attenuates diastolic Ca(2+) release in the aged murine heart that may prove useful in preventing cardiac dysfunction.

Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy

The Frank-Starling mechanism, whereby increased diastolic filling leads to increased cardiac output, depends on increasing the sarcomere length (Ls) of cardiomyocytes. Ventricular stiffness increases with advancing age, yet it remains unclear how such changes in compliance impact sarcomere dynamics in the intact heart. We developed an isolated murine heart preparation to monitor Ls as a function of left ventricular pressure and tested the hypothesis that sarcomere lengthening in response to ventricular filling is impaired with advanced age. Mouse hearts isolated from young (3-6 mo) and aged (24-28 mo) C57BL/6 mice were perfused via the aorta under Ca(2+)-free conditions with the left ventricle cannulated to control filling pressure. Two-photon imaging of 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium fluorescence was used to monitor t-tubule striations and obtain passive Ls between pressures of 0 and 40 mmHg. Ls values (in μm, aged vs. young, respectively) were 2.02 ± 0.04 versus 2.01 ± 0.02 at 0 mmHg, 2.13 ± 0.04 versus 2.23 ± 0.02 at 5 mmHg, 2.21 ± 0.03 versus 2.27 ± 0.03 at 10 mmHg, and 2.28 ± 0.02 versus 2.36 ± 0.01 at 40 mmHg, indicative of impaired sarcomere lengthening in aged hearts. Atomic force microscopy nanoindentation revealed that intact cardiomyocytes enzymatically isolated from aged hearts had increased stiffness compared with those of young hearts (elastic modulus: aged, 41.9 ± 5.8 kPa vs. young, 18.6 ± 3.3 kPa; P = 0.006). Impaired sarcomere lengthening during left ventricular filling may contribute to cardiac dysfunction with advancing age by attenuating the Frank-Starling mechanism and reducing stroke volume.

Elevated Ca2+ transients and increased myofibrillar power generation cause cardiac hypercontractility in a model of Noonan syndrome with multiple lentigines

Noonan syndrome with multiple lentigines (NSML) is primarily caused by mutations in the nonreceptor protein tyrosine phosphatase SHP2 and associated with congenital heart disease in the form of pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Our goal was to elucidate the cellular mechanisms underlying the development of HCM caused by the Q510E mutation in SHP2. NSML patients carrying this mutation suffer from a particularly severe form of HCM. Drawing parallels to other, more common forms of HCM, we hypothesized that altered Ca(2+) homeostasis and/or sarcomeric mechanical properties play key roles in the pathomechanism. We used transgenic mice with cardiomyocyte-specific expression of Q510E-SHP2 starting before birth. Mice develop neonatal onset HCM with increased ejection fraction and fractional shortening at 4-6 wk of age. To assess Ca(2+) handling, isolated cardiomyocytes were loaded with fluo-4. Q510E-SHP2 expression increased Ca(2+) transient amplitudes during excitation-contraction coupling and increased sarcoplasmic reticulum Ca(2+) content concurrent with increased expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In skinned cardiomyocyte preparations from Q510E-SHP2 mice, force-velocity relationships and power-load curves were shifted upward. The peak power-generating capacity was increased approximately twofold. Transmission electron microscopy revealed that the relative intracellular area occupied by sarcomeres was increased in Q510E-SHP2 cardiomyocytes. Triton X-100-based myofiber purification showed that Q510E-SHP2 increased the amount of sarcomeric proteins assembled into myofibers. In summary, Q510E-SHP2 expression leads to enhanced contractile performance early in disease progression by augmenting intracellular Ca(2+) cycling and increasing the number of power-generating sarcomeres. This gives important new insights into the cellular pathomechanisms of Q510E-SHP2-associated HCM.