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

The publisher's version of this article may be found at http://circres.ahajournals.org/cgi/content/abstract/90/11/1150?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1049671889562_931&stored_search=&FIRSTINDEX=0&volume=90&firstpage=1150&search_url=http%3A%2F%2Fcircres.ahajournals.org%2Fcgi%2Fsearch&journalMyocardial performance is likely affected by the relative expression of the two myosin heavy chain (MyHC) isoforms, namely {alpha}-MyHC and ß-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 {alpha}-MyHC. To this end, power output was measured in rat cardiac myocyte fragments that expressed {approx}12% {alpha}-MyHC and in myocyte fragments that expressed {approx}0% {alpha}-MyHC, as determined in the same cells by SDS-PAGE analysis after mechanical experiments. Myocyte fragments expressing {approx}12% {alpha}-MyHC developed {approx}52% greater peak normalized power output than myocyte fragments expressing {approx}0% {alpha}-MyHC. These results indicate that small amounts of {alpha}-MyHC expression significantly augment myocyte power output.This work was supported by National Heart, Lung, and Blood Institute Grant HL-57852 (K.S.M.) and a predoctoral fellowship granted by the Heartland Affiliate of the American Heart Association (T.J.H.)

What role does phosphorylation of cardiac troponin I play in elevating cardiac contractility following adrenergic stimulation?

Abstract only availableMyocardial performance is enhanced when adrenergic receptors are stimulated by catecholamines. The enhanced performance is mediated by cAMP dependent protein kinase A (PKA), which phosphorylates several proteins within the cardiac myocyte including cardiac troponin I (cTnI) and myosin binding protein-C (MyBP-C). Phosphorylation of these two proteins by PKA appears to mediate inotropic effects of adrenergic stimulation by directly modulating the rate of cross-bridge cycling. For instance, phosphorylation of cTnI and MyBP-C by PKA increased the power generating capacity of single permeabilized cardiac myocyte preparations (Herron, Korte, McDonald Circ Res 89;1184-1190:2001). It is unknown whether phosphorylation of cTnI or MyBP-C alone or phosphorylation of both proteins is necessary to increase power. The purpose of this study was to develop a methodology to test the relative importance of the two PKA phosphorylation sites on cTnI (serine 23 and 24) in mediating the PKA induced increase in myocyte power output. For these experiments, serines 23 and 24 of rat cTnI cDNA were mutated to alanines using site directed mutagenesis. Next, mutated cTnI (cTnI23/24) was expressed in E. coli bacteria and purified using ion exchange chromatography. Mutated cTnI (cTnI23/24) was then complexed with purified cTnT and cTnI and this whole troponin (cTn) complex was exchanged for endogenous cTn in permeabilized cardiac myocytes overnight using a cTn exchange buffer (20 mM Imidazole, 200 mM KCl, 5 mM EGTA, 5 mM MgCl2, 1 mM DTT). The extent of cTn exchange was assessed by quantifying the amount of PKA-induced phosphate incorporation. We observed only a partial reduction in PKA-induced phosphate incorporation following exhange of cTnI23/24, implicating only a partial Tn exchange using these conditions. We are currently seeking to increase the extent of cTnI23/24 exchange after which individual myocyte preparations will be mounted between a force transducer and position motor and myocyte power output generating capacity will be measured before and after PKA induced phosphorylation of myofilament proteins. These experiments will directly assess whether phosphorylation of cTnI by PKA is necessary to elevate myocyte power output.Life Sciences Undergraduate Research Opportunity Progra

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

Power Output Is Increased After Phosphorylation of Myofibrillar Proteins in Rat Skinned Cardiac Myocytes

This work was supported by American Heart Association Beginning Grant-in-Aid 9914291 and NIH Grant HL57852.The publisher's version may be found at http://circres.ahajournals.org/cgi/content/full/89/12/1184ß-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca2+ sensitivity of force. PKA also significantly increased both maximal force (25.4±8.3 versus 31.6±11.3 µN [P<0.001, n=12]) and peak absolute power output (2.48±1.33 versus 3.38±1.52 µW/mg [P<0.05, n=5]) during maximal Ca2+ activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased {approx}20% during maximal Ca2+ activations (n=5) and {approx}33% during half-maximal Ca2+ activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output-generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after ß-adrenergic stimulation

Role of cardiac troponin I phosphorylation in cardiac function: From molecule to mouse

Abstract only availableThe regulation of cardiac muscle contraction involves the interplay between a variety of molecules on the thick and thin filaments. One important regulatory molecule is troponin, which consists of three subunits, troponin C (TnC) that binds calcium, troponin T (TnT) that binds tropomyosin, and troponin I (TnI) that binds actin and tends to inhibit contraction. Following muscle excitation, cytoplasmic calcium rises and binds TnC, which causes a conformational change in TnI that reduces its affinity for actin; this, in turn, allows TnT and tropomyosin to shift positions revealing myosin binding sites on actin, leading to muscle contraction. Interestingly, cardiac troponin I (cTnI) has several phosphorylation sites, which are known to modulate this regulatory process. For example, phosphorylation of serines 23 and 24 on cTnI by protein kinase A (PKA) is known to decrease the calcium binding affinity of cardiac TnC and, thus, thought to speed muscle relaxation. On the other hand, phosphorylation of cTnI on serines 43 and 45 and threonine 144 by protein kinase C (PKC) decreases both force production and calcium sensitivity of force and is thought to contribute to depressed ventricular function in failing hearts. In this study we investigated the effects of chronic cTnI phosphorylation on cardiac function from transgenic animals in which either PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both the PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid to mimic phosphorylation. Left ventricular cardiac myocytes from PP transgenic mice exhibited less calcium sensitivity of force while myocytes from All-P transgenic mice exhibited decreased maximal force, decreased calcium sensitivity of force, and decreased power output, implicating a dominate role of PKC phosphorylation sites on myofilament function. Consistent with these single myocyte studies, left ventricular power output also was depressed in All-P mice compared to both WT and PP transgenic ventricles. We next tested the hypothesis that PP transgenic mice would engage in greater voluntary running compared to WT and All-P transgenic animals. In contrast to this idea, WT and All-P mice ran ~3- and ~4-fold more than the PP transgenic mouse, respectively. Overall, these results indicate that PKC phosphorylation of cTnI plays a dominant role in depressing contractility and may contribute to the maladaptive behavior.NIH grant to K.S. McDonal

Increasing the availability and utilization of reliable data on population micronutrient (MN) status globally: the MN Data Generation Initiative.

Micronutrient (MN) deficiencies can produce a broad array of adverse health and functional outcomes. Young, preschool children and women of reproductive age in low- and middle-income countries are most affected by these deficiencies, but the true magnitude of the problems and their related disease burdens remain uncertain because of the dearth of reliable biomarker information on population MN status. The reasons for this lack of information include a limited understanding by policy makers of the importance of MNs for human health and the usefulness of information on MN status for program planning and management; insufficient professional capacity to advocate for this information and design and implement related MN status surveys; high costs and logistical constraints involved in specimen collection, transport, storage, and laboratory analyses; poor access to adequately equipped and staffed laboratories to complete the analyses reliably; and inadequate capacity to interpret and apply this information for public health program design and evaluation. This report describes the current situation with regard to data availability, the reasons for the lack of relevant information, and the steps needed to correct this situation, including implementation of a multi-component MN Data Generation Initiative to advocate for critical data collection and provide related technical assistance, laboratory services, professional training, and financial support

Recruitment of young women to a trial of chlamydia screening – as easy as it sounds?

BACKGROUND: Recruiting to trials is complex and difficult. The Prevention of Pelvic Infection (POPI) trial aims to see if screening women for chlamydia and treating those found to be infected reduces the incidence of pelvic inflammatory disease in the following twelve months. It focuses on young, sexually active, multiethnic, mainly inner city, female students. The main aim of this paper is to describe our recruitment methods. Secondary aims in two small subgroups, are to compare characteristics of women recruited with those not recruited, and to explore participants' understanding of when their samples would be tested for chlamydia. METHODS: Women students attending lectures or in common rooms at 22 universities and further education colleges were recruited by female research assistants working in pairs. Participants were asked to complete a questionnaire on sexual health and to provide self-taken vaginal swabs. In addition, during 3 recruitment sessions, a female medical student asked non-participants to complete a brief anonymous questionnaire on reasons for not taking part. Finally another female medical student contacted 40 consecutive participants within a month of recruitment and asked if they understood that their samples might not be tested for a year. RESULTS: With enormous effort over 2 years we recruited 2526 women. A survey of 61 non-responders showed only 18 (30%) were eligible to take part (age <28, been sexually active and not been tested for chlamydia in the past 3 months). Eligible non-responders were of similar age to the 35 responders in the same recruitment sessions, but more likely to be from ethnic minority groups (67% 12/18 versus 29% 10/35 p < 0.01). Email and telephone contact with 35/40 (88%) of consecutive participants showed only two (6%) did not understand that their specimen might not be tested for chlamydia for a year. Thirty participants (85%) could name one or more possible consequences of untreated chlamydia infection. CONCLUSION: As in other studies, a key to attaining recruitment targets was the enthusiasm of the research team. Minority ethnic groups were probably under-represented, but understanding of participants was good

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF

Impacts of local human activities on the Antarctic environment

We review the scientific literature, especially from the past decade, on the impacts of human activities on the Antarctic environment. A range of impacts has been identified at a variety of spatial and temporal scales. Chemical contamination and sewage disposal on the continent have been found to be long-lived. Contemporary sewage management practices at many coastal stations are insufficient to prevent local contamination but no introduction of non-indigenous organisms through this route has yet been demonstrated. Human activities, particularly construction and transport, have led to disturbances of flora and fauna. A small number of non-indigenous plant and animal species has become established, mostly on the northern Antarctic Peninsula and southern archipelagos of the Scotia Arc. There is little indication of recovery of overexploited fish stocks, and ramifications of fishing activity oil bycatch species and the ecosystem could also be far-reaching. The Antarctic Treaty System and its instruments, in particular the Convention for the Conservation of Antarctic Marine Living Resources and the Environmental Protocol, provide a framework within which management of human activities take place. In the face of the continuing expansion of human activities in Antarctica, a more effective implementation of a wide range of measures is essential, in order to ensure comprehensive protection of the Antarctic environment, including its intrinsic, wilderness and scientific values which remains a fundamental principle of the Antarctic Treaty System. These measures include effective environmental impact assessments, long-term monitoring, mitigation measures for non-indigenous species, ecosystem-based management of living resources, and increased regulation of National Antarctic Programmes and tourism activities