- Email: [email protected]
Structural characterization and expression of the murine cardiac troponin C gene
J Mol
Cell
Cardiol
21 (Supplement
II) (1989)
187
STRUCTURAL CHARACTERIZATION AND EXPRESSION OF THE MURINE CARDIAC TROPONIN C GENE. Michael S. Parmacek and Jeffrey M. Leiden. Howard Hughes Medical Institute and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan. Troponin C (cTnC) is the calcium binding subunit of the thin filament which regulates excitationcontraction coupling. In order to better study the transcriptional regulation of this gene as well as to understand the structural basis of troponin C function, we have isolated and characterized the cTnC cDNA and gene. The murine cardiac troponin C cDNA was isolated directly from total cardiac RNA by the technique of polymerase chain reaction (PCR). The murine cTnC cDNA encodes a protein of 161 amino acids which has been highly conserved during evolution as the deduced amino acid sequence is 98% (158/161 a.a.) homologous to chicken cTnC and 99% (160/161 a.a) homologous to human cTnC. Primer extension analysis revealed that the start of cTnC transcription is located 35 base pairs 5’ of the initiation codon. Southern blot analyses demonstrated that the cTnC gene is a member of a multi-gene family. Northern blot analyses showed that cTnC is expressed in both cardiac tissue and slow skeletal muscle (soleus), but is not expressed in fast skeletal muscle (EDL and anterior tibialis). Moreover, cTnC was not sianificantlv _ exoressed in neonatal or adult brain..kidnev. . . luna.-, liver and testis. The . cTnC cDNA was used as a hybridization probe to screen 1 x Id6 recombinant clones from a murine EMBL3 genomic library. Thirteen cTnC clones were identified and structurally characterized. One clone (gTnC7) was found to encode the entire gene and was subjected to DNA sequence analysis. The cTnC gene is approximately 4 kilobases in length and is composed of 6 exons and 5 introns. Important regulatory regions of the gene have been identified and will be described.
188
NUMERICAL ANALYSIS OF PHYSIOLOGIC FUNCTION CURVES. R. K. Helbing and P.B. Taylor. Department of Physics and Biological Sciences, Univ. of Windsor, Windsor, Ontario, Canada N9B 3P4. The analvsis of function curves. is essential in the description and quantification of biological responses: The use of microcomputers has extended the ability to control experiments and store data but the inherent difficulties in data analysis still remains; The major problems are associated with a lack of symmetery, complex curvlinear regions within the function and the possibility that the chacteristics of the function are amplitude dependent. In general, two approaches have evolved. Linear extrapolation to the baseline from the slope of either the rising or falling phase and more recently the use of exponential models. Both methods have important limitations. Extrapolation can underestimate the time base because it ignores the curvlinear portions at the beginning and the end of the function. The exponential models assume that the curvelinear portions follow a true exponential function. In addition, both methods are still very sensitive to uncontrolled changes in the amplitude of the signal. We have developed a procedure and a computer program that uses an arc cosine transformation. This clearly isolates the beginning and the The orocedure end of the function and eliminates variations due to amolitude chanae. has been successfully applied to ventricular pressure-and twitch tension curves in isolated bundles of muscle(Supported by Ontario Heart and Stroke Foundation and NSERC Canada).
189CONTRHCTILE RESERVE OF RIGHT AND LEFT VENTRICLES AFTER ISCHEMIC ARREST AND CARDIOPLEGIA. J.S. Juqqi, A.M. Yousof and Hani J. Shuhaiber. Fat. of Med.. Klmait Univ.. Kuwait (A. Gulf). Contractile reserve (CR) of right (RV) and left (LV) ventricles of the isolated perfused rat heart was studied from the Frank-Starling (FS) (heterometric reserve, HR) and interval-force (inotrooic reserve. IR) relationshios. Hearts were subiected to either one hour of unprotected ischemia (34°C) or protected with hypothermia (ZO'C) and hypothermic cardioplegia (STH: St. Thomas' Hosoital: GPN: olucose-ootassium-nifedioine) durino the ischemic oeriod. t& was quantified by fitting poly&nial functions to FS curves. IR-was quantified 'by the technique of post-stimulation potentiation. The results of these studies are summarized: Heterotnetric Reserve Inotropic Reserve Iz (X. Control), I, I I I , a , , I (%? r&ntrol ) meansfSE (n=6) Rv R\I ISCH. 34°C Z.0ts.S 26;&$ 63.5f22 32.0+7? * P< 0.05
ISCH. 20°C
74.4ti.i
73.6&
94.525.0
98.5f8.8
STH.
88.3f6.7
93.3t6.2
65.6f16.6
53.ti5?
20°C
**
PC 0.01
** P< 0.001 GPN. 20°C 92.5ti.l 89.5S.9 47.3f7.i 73.2k16.9 The results indicate: (i) severe depression of CR by plain ischemia, (ii) improvement of IR by hypothermia and depression by cardioplegia, and (iii) improvement of HR by cardioplegia. Supported by Kuwait University research grants MY021 and RAO30. S.63
Cell
Cardiol
21 (Supplement
II) (1989)
187
STRUCTURAL CHARACTERIZATION AND EXPRESSION OF THE MURINE CARDIAC TROPONIN C GENE. Michael S. Parmacek and Jeffrey M. Leiden. Howard Hughes Medical Institute and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan. Troponin C (cTnC) is the calcium binding subunit of the thin filament which regulates excitationcontraction coupling. In order to better study the transcriptional regulation of this gene as well as to understand the structural basis of troponin C function, we have isolated and characterized the cTnC cDNA and gene. The murine cardiac troponin C cDNA was isolated directly from total cardiac RNA by the technique of polymerase chain reaction (PCR). The murine cTnC cDNA encodes a protein of 161 amino acids which has been highly conserved during evolution as the deduced amino acid sequence is 98% (158/161 a.a.) homologous to chicken cTnC and 99% (160/161 a.a) homologous to human cTnC. Primer extension analysis revealed that the start of cTnC transcription is located 35 base pairs 5’ of the initiation codon. Southern blot analyses demonstrated that the cTnC gene is a member of a multi-gene family. Northern blot analyses showed that cTnC is expressed in both cardiac tissue and slow skeletal muscle (soleus), but is not expressed in fast skeletal muscle (EDL and anterior tibialis). Moreover, cTnC was not sianificantlv _ exoressed in neonatal or adult brain..kidnev. . . luna.-, liver and testis. The . cTnC cDNA was used as a hybridization probe to screen 1 x Id6 recombinant clones from a murine EMBL3 genomic library. Thirteen cTnC clones were identified and structurally characterized. One clone (gTnC7) was found to encode the entire gene and was subjected to DNA sequence analysis. The cTnC gene is approximately 4 kilobases in length and is composed of 6 exons and 5 introns. Important regulatory regions of the gene have been identified and will be described.
188
NUMERICAL ANALYSIS OF PHYSIOLOGIC FUNCTION CURVES. R. K. Helbing and P.B. Taylor. Department of Physics and Biological Sciences, Univ. of Windsor, Windsor, Ontario, Canada N9B 3P4. The analvsis of function curves. is essential in the description and quantification of biological responses: The use of microcomputers has extended the ability to control experiments and store data but the inherent difficulties in data analysis still remains; The major problems are associated with a lack of symmetery, complex curvlinear regions within the function and the possibility that the chacteristics of the function are amplitude dependent. In general, two approaches have evolved. Linear extrapolation to the baseline from the slope of either the rising or falling phase and more recently the use of exponential models. Both methods have important limitations. Extrapolation can underestimate the time base because it ignores the curvlinear portions at the beginning and the end of the function. The exponential models assume that the curvelinear portions follow a true exponential function. In addition, both methods are still very sensitive to uncontrolled changes in the amplitude of the signal. We have developed a procedure and a computer program that uses an arc cosine transformation. This clearly isolates the beginning and the The orocedure end of the function and eliminates variations due to amolitude chanae. has been successfully applied to ventricular pressure-and twitch tension curves in isolated bundles of muscle(Supported by Ontario Heart and Stroke Foundation and NSERC Canada).
189CONTRHCTILE RESERVE OF RIGHT AND LEFT VENTRICLES AFTER ISCHEMIC ARREST AND CARDIOPLEGIA. J.S. Juqqi, A.M. Yousof and Hani J. Shuhaiber. Fat. of Med.. Klmait Univ.. Kuwait (A. Gulf). Contractile reserve (CR) of right (RV) and left (LV) ventricles of the isolated perfused rat heart was studied from the Frank-Starling (FS) (heterometric reserve, HR) and interval-force (inotrooic reserve. IR) relationshios. Hearts were subiected to either one hour of unprotected ischemia (34°C) or protected with hypothermia (ZO'C) and hypothermic cardioplegia (STH: St. Thomas' Hosoital: GPN: olucose-ootassium-nifedioine) durino the ischemic oeriod. t& was quantified by fitting poly&nial functions to FS curves. IR-was quantified 'by the technique of post-stimulation potentiation. The results of these studies are summarized: Heterotnetric Reserve Inotropic Reserve Iz (X. Control), I, I I I , a , , I (%? r&ntrol ) meansfSE (n=6) Rv R\I ISCH. 34°C Z.0ts.S 26;&$ 63.5f22 32.0+7? * P< 0.05
ISCH. 20°C
74.4ti.i
73.6&
94.525.0
98.5f8.8
STH.
88.3f6.7
93.3t6.2
65.6f16.6
53.ti5?
20°C
**
PC 0.01
** P< 0.001 GPN. 20°C 92.5ti.l 89.5S.9 47.3f7.i 73.2k16.9 The results indicate: (i) severe depression of CR by plain ischemia, (ii) improvement of IR by hypothermia and depression by cardioplegia, and (iii) improvement of HR by cardioplegia. Supported by Kuwait University research grants MY021 and RAO30. S.63