Paul Paolini

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.

The Antidiabetic Agent Rosiglitazone Upregulates SERCA2 and Enhances TNF-α- and LPS-Induced NF-κB-Dependent Transcription and TNF-α- Induced IL-6 Secretion in Ventricular Myocytes

Positive hemodynamic effects of the antidiabetic agent rosiglitazone on perfused whole hearts have recently been described, but the mechanisms regulating these effects are not well understood. This study reports the effects of rosiglitazone on calcium regulation in isolated neonatal rat ventricular myocytes by measurement of Ca2+ transient decay rates and SERCA2 gene expression, and shows that rosiglitazone enhances known cardioprotective signaling pathways. Myocyte treatment with 10 μmol/L rosiglitazone accelerated Ca2+ transient decay rates by ñ30%, enhanced SERCA2 mRNA levels by ñ1.5-fold and SERCA2 production by ñ3-fold. Rosiglitazone treatment (1, 5, and 10 μmol/L) also led to a dose-dependent increase (ñ1.2-1.5-fold) in SERCA2 promoter activity. Comparable levels of cardiac SERCA overexpression have been associated with physiologically relevant and compensatory effects in vivo. These data link thiazolidinedione-induced improvement in cardiac myocyte function to an upregulation of SERCA2 gene expression. Since NF-κB-dependent pathways, including the upregulation of IL-6 secretion, were shown to protect neonatal rat ventricular myocytes from apoptosis upon TNFα stimulation, additional experiments were designed to determine whether rosiglitazone enhances TNFα-induced NF-κB-dependent transcription and IL-6 secretion. Because the endotoxin stress response in ventricular myocytes involves the upregulation of TNFα, and the activation of NF-κB, the effects of rosiglitazone on lipopolysaccharide-induced NF-κB-dependent transcription were also investigated. Treatment of neonatal rat ventricular myocytes with 10 μmol/L rosiglitazone enhanced TNF-α- and lipopolysaccharide-induced NF-κB-dependent transcription by ñ1.8- and ñ1.4-fold respectively, and TNF-α-induced IL-6 secretion by ñ1.5-fold. Rosiglitazone had no significant effects on basal levels of NF-κB-dependent transcription and IL-6 secretion. Thus, cardioprotective effects of rosiglitazone may be partly mediated by NF-κB.

Digital data acquisition and analysis of striated muscle diffraction patterns with a direct memory access microprocessor system

A microprocessor based data acquisition computing system has been developed to examine dynamic changes in light diffraction patterns from single skeletal muscle fibers. A significant improvement in digital data acquisition rate compared to previous designs has been achieved by utilizing a fast dedicated analog to digital converter and direct memory access data storage. Diffraction patterns from muscle fibers are imaged onto a 256 element charge‐coupled device. The analog output of a full 256 point frame of data may be digitized and stored in 2.2 ms (128 point half frame in 1.1 ms) with a spatial resolution of up to 5.0 nm/sarcomere. This computing system can transfer up to 28 full frames of data as one continuous block directly between the CCD and memory leaving the CPU free for experimental control and closed‐loop processing. The computing system calibrates and analyzes diffraction data under sofware control for sarcomere length, dispersion, and peak intensity. The operation of this data acquisition comp...

Image processing techniques for assessing contractility in isolated neonatal cardiac myocytes

We describe a computational framework for the quantitative assessment of contractile responses of isolated neonatal cardiac myocytes. To the best of our knowledge, this is the first report on a practical and accessible method for the assessment of contractility in neonatal cardiocytes. The proposed methodology is comprised of digital video recording of the contracting cell, signal preparation, representation by polar Fourier descriptors, and contractility assessment. The different processing stages are variants of mathematically sound and computationally robust algorithms very well established in the scientific community. The described computational approach provides a comprehensive assessment of the neonatal cardiac myocyte contraction without the need of elaborate instrumentation. The versatility of the methodology allows it to be employed in determining myocyte contractility almost simultaneously with the acquisition of the Ca2+ transient and other correlates of cell contraction. The proposed methodology can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease models, transgeneity, or other common applications of neonatal cardiocytes.

Dual‐channel diffractometer utilizing linear image sensor charge‐coupled devices

A position‐sensitive dual‐channel light diffracometer has been developed to allow the real‐time monitoring of light diffraction patterns produced by the myofibrillar sarcomeres of single skeletal muscle fibers and small bundles of fibers during contraction. The diffractometer utilizes two linear image devices to permit the analysis of two or more diffraction lines. The sensors are highly sensitive (1.67×10−4 lux sec), lag‐free, self‐scanning 256‐element charge‐coupled devices driven by Schottky and standard TTL logic circuitry at a data transfer rate/element of about 20–400 kHz. Video output from the diffractometer can be displayed directly on an oscilloscope screen or can be connected to an ADC and minicomputer system so that diffraction spectra can be corrected for dark signal and element nonuniformities and displayed on‐line by the computer CRT. The computer‐evaluated spacing between diffraction lines provides an accurate measure of mean sarcomere length within the muscle, while line shape and width ar...

Developmental and extracellular matrix-remodeling processes in rosiglitazone-exposed neonatal rat cardiomyocytes

OBJECTIVE The objective of this study was to investigate the effects of rosiglitazone (Avandia(®)) on gene expression in neonatal rat ventricular myocytes. MATERIALS & METHODS Myocytes were exposed to rosiglitazone ex vivo. The two factors examined in the experiment were drug exposure (rosiglitazone and dimethyl sulfoxide vs dimethyl sulfoxide), and length of exposure to drug (½ h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h and 48 h). RESULTS Transcripts that were consistently expressed in response to the drug were identified. Cardiovascular system development, extracellular matrix and immune response are represented prominently among the significantly modified gene ontology terms. CONCLUSION Hmgcs2, Angptl4, Cpt1a, Cyp1b1, Ech1 and Nqo1 mRNAs were strongly upregulated in cells exposed to rosiglitazone. Enrichment of transcripts involved in cardiac muscle cell differentiation and the extracellular matrix provides a panel of biomarkers for further analysis in the context of adverse cardiac outcomes in humans. Original submitted 15 November 2013; Revision submitted 14 February 2014.

Calcium-induced structural changes in chemically skinned muscle fibers. Detection by optical diffractometry.

The intensity of the first order diffraction line produced by chemically skinned muscle fibers was detected by a self scanning photodiode array and minicomputer system. Line intensity was observed to decrease in fibers stretched to zero filament overlap when subjected to calcium-EGTA buffers in the physiological pCa range. Calcium dependent intensity decreases were not observed for myosin extracted fibers indicating that the thick filament proteins may be the source of the calcium effect seen in non-extracted fibers. These results can be interpreted in terms of calcium dependent effects on thick filament disordering which are not dependent upon cross bridge formation.

Calcium and pH-induced structural changes in skinned muscle fibers: Prevention by N-ethylmaleimide

Abstract Optical diffractometry was used to investigate structural changes in contractile proteins from chemically skinned muscle fibers. The intensity of the first order diffraction line decreased in response to calcium and alkaline pH. Fibers pre-treated with sulfhydryl blocking agents did not exhibit the calcium- or pH- induced intensity decreases. We suggest that the decreases in intensity result from structural changes in the thick filament. Optical diffractometry may be a useful technique for the investigation of conformational changes in myosin.

Contractility assessment in enzymatically isolated cardiomyocytes

The use of enzymatically isolated cardiac myocytes is ubiquitous in modern cardiovascular research. Parallels established between cardiomyocyte shortening responses and those of intact tissue make the cardiomyocyte an invaluable experimental model of cardiac function. Much of our understanding regarding the fundamental processes underlying heart function is owed to our increasing capabilities in single-cell stimulation and direct or indirect observation, as well as quantitative analysis of such cells. Of the many important mechanisms and functions that can be readily assessed in cardiomyocytes at all stages of development, contractility is the most representative and one of the most revealing. The purpose of this review is to provide a survey of various methodological approaches in the literature used to assess adult and neonatal cardiomyocyte contractility. The various methods employed to evaluate the contractile behavior of enzymatically isolated mammalian cardiac myocytes can be conveniently divided into two general categories—those employing optical (image)-based systems and those that use transducer-based technologies. This survey is by no means complete, but we have made an effort to include the most popular methods in terms of reliability and accessibility. These techniques are in constant evolution and hold great promise for the next generation of breakthrough studies in cell biology for the prevention, treatment, and cure of cardiovascular diseases.