Acknowledgements:
I would like to acknowledge and sincerely thank Dr. Jose Pinto, Dr. Diego Zorio, Dr. Adriano Martins, Dr. Igor Alabugin, Dr. Thomas Keller, Dr. Karen McGinnis,Dr. Hedi Mattoussi, Dr. Brian Miller, Omar Awan, Matthew D’Alessandro, Jamie Johnston, David Gonzalez-Martinez andBrittany Griffin their help, guidance and support of my honors thesis project.
I would like to acknowledge and thank the founders of the Bess H. Ward research award grant.
Abstract:
Quantitative polymerase chain reaction (qPCR) is an experimental technique used to determine the initial quantity of a targeted sequence in a sample, compared to a control group. The specific aim of this paper is to determine the gene expression level of various heart proteins in mice with dilated cardiomyopathy (DCM), compared to wildtype (WT) mice. It is hypothesized that various proteins will either be upregulated or downregulated, depending on their function in the myocyte. The proteins being tested are: atrial natriuretic peptide, brain natriuretic peptide, collagen I and III, triadin, S100A, sarco endoplasmic reticulum Ca2+ ATPase, phospholamban, calsequestrin, and sodium/calcium exchanger. Quantitative polymerase chain reaction experiments were performed on RNA from the left ventricle, which was extracted using RNA-bee (Tel-Test Bulletin, Catalog number CS-104B). Reverse transcription was performed using iSCRIPT Select cDNA synthesis kit (BioRad catalog number 170-8896). The solutions used for qPCR consisted of SYBR green (Quanta Biosciences, catalog number 95056-100), forward primer, reverse primer, cDNA, nuclease free water. Optimal primer concentration was determined and primer specificity was tested by blasting all primers on PubMed, and creating melting curves for each primer. The results for this paper were inconclusive as there was a problem with the mice expression their genetic mutation which resulted in DCM.
List of Tables:
Table 1: Primer name, sequence and melting temperature. (Page 12)
Table 2: Blast results of all primers. (Page 15)
List of Figures:
Figure 1A: Head, neck, and tail domains of one myosin protein. (Page 3)
Figure 1B: The HMM and LMM fragments. (Page 3)
Figure 1C: The S1 and S1 sub fragments of the HMM fragment. (Page 4)
Figure 2: Myosin proteins together to form a “bipolar” filament with head domains together and both ends. (Page 4)
Figure 2: Cross bridge cycle of actin and myosin in the presence of ATP. (Page 5)
Figure 3: Sarcomere contracted and relaxed. (Page 6)
Figure 5: Tm and Tn on an actin filament. (Page 6)
Figure 6: T-tubule entering into the cell. (Page 7)
Figure 7: Nanodrop reading from extracted RNA from left ventricle tissue. (Page 14)
Figure 8: Nanodrop reading from cDNA reverse transcribed from the extracted RNA in figure 7. (Page 14)
Figure 9: Melt Curve of atrial natriuretic peptide at an initial primer concentration of 10uM. (Page 17)
Figure 10: Melt Curve of brain natriuretic peptide at an initial primer concentration of 10uM. (Page 17)
Figure 11: Melt Curve of collagen I at an initial primer concentration of 10uM. (Page 18)
Figure 12: Melt Curve of collagen III at an initial primer concentration of 10uM. (Page 18)
Figure 13: Melt Curve of Triadin at an initial primer concentration of 10uM. (Page 19)
Figure 14: Melt Curve of sarco-endoplasmic reticulum Ca2+ATpase at an initial primer concentration of 10uM. (Page 19)
Figure 15: Melt Curve of S100A at an initial primer concentration of 10uM. (Page 20)
Figure 16: Melt Curve of calsequestrin at an initial primer concentration of 10uM. (Page 20)
Figure 17: Melt Curve of cyclophilin at an initial primer concentration of 100uM. (Page 21)
Figure 18: Melt Curve of βactin at an initial primer concentration of 10uM. (Page 21)
Figure 19: Melt Curve of Sodium Calcium Exchanger at an initial primer concentration of 10uM. (Page 22)
Figure 20: Melt Curve of phospholamban at an initial primer concentration of 10uM. (Page 22)
Figure 21A: Melt Curve of cyclophilin at an initial primer concentration of 100uM. (Page 23)
Figure 21B: Melt Curve of cyclophilin at an initial primer concentration of 10uM. (Page 23)
Figure 22A: Melt Curve of sarcoglycan α at an initial primer concentration of 100uM. (Page 24)
Figure 22B: Melt Curve of sarcoglycan α at an initial primer concentration of 10uM. (Page 24)
Background:
“The heart contracts without interruption about 3 million times a year, or a fifth of a billion times in a lifetime.” (Lodish, et al.).
Cardiac Muscle Physiology:
Muscle contraction occurs when myosin and actin interact with one another. The interaction of myosin and actin is regulated by troponin and tropomyosin, which in turn is regulated by calcium (Ca2+). (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014).
Myosin is a motor protein found in the cell. There are several different classes of myosin, however all myosin proteins share one common property, they all use the energy released in ATP hydrolysis for mechanical work in the cell. Myosin II uses this energy for muscle contraction. (Lodish, et al.)
Myosin is made up of six polypeptides, two of the polypeptides are identical and have a high molecular weight, and thus they are called “myosin heavy chains.” Each myosin heavy chain has three domains: head, neck and tail, as seen in Figure 1A.The last four polypeptides that make up myosin have a lower molecular weight and are thus called the “myosin light chains”, there are regulatory and essential light chains. (Lodish, et al.)
Myosin II can be further classified into the heavy meromyosin (HMM) fragment and the light meromyosin (LMM) fragment, as seen in Figure 1B. The HMM fragment consists of the S1 and S2 sub fragments, as seen in Figure 1C. The head domain consists of the actin binding sites, which allow myosin-actin interactions, as well as the ATPase activity. The neck domain consists of the myosin light chains, both regulatory and essential, and the tail domain consists of the two myosin heavy chains in a coil-coil interaction. (Lodish, et al.)
Figure 1B:The HMM and LMM fragments. Source: (Lodish, et al.), labels edited for paper.
Figure 1C: The S1 and S1 sub fragments of the HMM fragment. Source: (Lodish, et al.), labels edited for paper.
As seen in Figure 2, many different myosin’s cluster together to form a bipolar filament.
Figure 2: Myosin proteins together to form a “bipolar” filament with head domains together and both ends. Source:
Actin assembles into a polymer, F-actin that is made up of many linear G-actin monomers, can be seen in Figure 3 below. Actin has functionally distinct ends, (+) end and (-) end. The difference in the ends of actin is that actin monomers favor the addition to the (+) end over the (-) end. (Lodish, et al.)
Cross-Bridge Cycle:
When no ATP is present, the myosin head is tightly bound to actin. When one ATP molecule binds to the myosin head domain, the myosin loses affinity for actin, thus myosin and actin are no longer bound. The myosin head then hydrolyzes the ATP to ADP and Pi, which rotates the myosin head into a “cocked state.” In this cocked state, the myosin head stores the energy released during ATP hydrolysis. Also in this cocked state, the myosin head binds to actin. Upon the release of ADP and Pi the stored energy is also released, resulting in a “power stroke” which essentially moves the myosin head along the actin. The exponential repetition of this cycle generates muscle contraction. This interaction that occurs can also be known as a “cross-bridge cycle”, shown in Figure 3. (Lodish, et al.)
Figure 5: Cross bridge cycle of actin and myosin in the presence of ATP. Source:
In each cardiac muscle cell, there are many myofibrils that are composed of a repeating array of sarcomeres, which are the basic unit of contraction. Each sarcomere is made up of the thick and thin filaments, as shown in Figure 4. The thick filaments are composed of the myosin II bipolar filaments. The thin filaments are composed of the actin filaments which are assembled with their (+) ends orientated in the same direction, towards the Z disk. During the cross-bridge cycle,the myosin heads move toward the Z-disk and essentially shorten the sarcomere and thus the muscle contracts. (Lodish, et al.)
Figure 6: Sarcomere contracted and relaxed. Source:
Actin and myosin are not always able to interact with one another. Their interaction is regulated by the tropomyosin (Tm) and troponin (Tn) complex. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). When Tm is bound to actin, it blocks the myosin binding sites, thus inhibiting muscle contraction, as seen in Figure 5.(Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). The Tn protein is made up of three subunits: Troponin I (TnI), Troponin C, (TnC), and troponin T, (TnT). (Willott, et al., 2009) Each subunit plays a specific role in muscle contraction and relaxation. Troponin I is an ATPase inhibitory protein, TnC is a Ca2+ binding protein, and TnT binds the entire Tn complex to Tm. (Willott, et al., 2009) The Tm and Tn inhibition can be removed in the presence of calcium (Ca2+). (Lodish, et al.)
Figure 5: Tm and Tn on an actin filament. Source:
The C- and N- domains of TnC both consist of EF hand motifs. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). The C-domain has two binding pockets for a cation, while the N-domain has only one binding pocket. It has been shown that the C-domain binding sites are always occupied with either Ca2+ or Mg2+, depending on the concentration of each cation in the cytosol. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). Under the resting concentration of 1mM Mg2+, the N-domain binding site has been shown to have a 33-44% saturation with Mg2+. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014)
However, when the concentration of Ca2+ in the cytosol is increased past its resting concentration of below 0.1μM (Lodish, et al.) to 10μM during an action potential, (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014) Ca2+ binds to the N-domain binding site, which in turn causes a conformational change of TnI and Tm, which releases the Tm actin-myosin inhibition and allows myosin to bind to actin. (Kalyva, Parthenakis, Marketou, Kontaraki, & Vardas, 2014). Also, the Ca2+ binding to TnC relieves the TnI inhibition of ATPase activity. (Willott, et al., 2009) Therefore, muscle contraction occurs.
Excitation-Contraction Coupling:
During an action potential, Ca2+ travels from the sarcoplasmic reticulum (SR), where it is bound to calsequestrin (CSQ) (Liew & Dzau, 2004) to the myocyte cytosol via proteins called ryanodine receptors (RyR). (Zima & Terentyev, 2013) The RyR channels only release the SR Ca2+ when they are activated, which happens as a result of a small Ca2+ current via the L-type channels on the T-tubules on the sarcolemma, as shown in Figure 6.(Zima & Terentyev, 2013)
Thus, the Ca2+ release from the RyR channels is referred to as Ca2+ induced Ca2+ release. (Zima & Terentyev, 2013). Once in the cytosol, the Ca2+ binds to TnC, and muscle contraction occurs. Muscle relaxation occurs when the Ca2+ is removed from the cytosol. This Ca2+ removal can happen via the protein sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2A) which transports Ca2+ back into the SR, or the Ca2+ can exit the cell via plasma membrane calcium ATPase (PMCA) or the sodium/calcium exchanger (NCX). (Liew & Dzau, 2004). SERCA2A is regulated by the protein phospholamban (PLB). If PLB is phosphorylated by protein kinase A (PKA), it will disassociate from SERCA2A, and thus the inhibition is removed and SERCA2A can reuptake CA2+ into the SR. (Liew & Dzau, 2004).
Molecular Markers for Heart Disease:
The protein atrial natriuretic peptide (ANP) functions to maintain cardiac homeostasis. This vasodilator protein is released in the heart ventricle in response to high blood volume and cardiac hypertrophy. A similar protein, brain natriuretic peptide (BNP) has also been shown to have an increased synthesis during cardiac hypertrophy (Gardner, 2003). Other proteins that has been shown to have an altered expression level during heart disease are Collagen Type I and III (Col I and Col III, respectively)(Pauschinger, Knopf, Petschauer, & Doerner, 1999). Collagen fibers can be described as proteins that provide “structural integrity, mechanical strength, and resilience.” (Lodish, et al.) This altered expression level may mean many things for the heart muscle, as each type of Collagen has its own properties. For example, Collagen I is mainly in stiff tissues, while Collagen III is mainly found in more elastic tissues.Another important protein is S100A, which is a member of the S100 protein family. S100A is a Ca2+ binding protein that interacts with SERCA2A and PLB, and it has been shown that an increase in S100A1 results in an increase in SERCA2A activity. (Duarte-Costa, Castro-Ferreira, Neves, & Leite-Moreira, 2014). One final important protein that may have a change in expression during heart disease is the protein triadin (trdn). Triadin (trdn) functions to connect RyR to the Ca2+ buffering protein, CSQ. (Chopra & Knollmann, 2013).
Introduction:
The heart disease dilated cardiomyopathy (DCM) is a disease that affects the contraction of the heart muscle. This disease often results in an enlargement or dilation of the left ventricle, hence the name dilated cardiomyopathy. (Hershberger, Hedges, & Morales, 2013). During this disease state, the heart will try to maintain asymptomatic and perform normal cardiac functions (Liew & Dzau, 2004). This may result in an altered expression level of certain proteins in the heart, compared to a control wildtype (WT) heart that does not have a mutation.
Specific Aim:
The specific aim of this paper is to determine the gene expression level of various heart proteins in mice with a DCM disease, compared to WT mice.To quantify the levels of gene expression, quantitative polymerase chain reaction (qPCR) was used. The hypothesis to be tested is that the diseased heart will have an upregulation or downregulation of various heart proteins in response to the DCM disease.
Quantitative polymerase chain reaction:
Quantitative polymerase chain reaction (qPCR) is an experimental technique where specific regions of DNA or complementary DNA (cDNA, DNA synthesized from RNA) are amplified many thousand fold. The specific regions that are amplified are determined by forward and reverse primers that are added to the reaction solution. These primers are designed to bind to a specific sequence that corresponds to a specific gene, and thus only that gene sequence is amplified. (Life Technologies).
The qPCR experiments consist of three steps, which are: denaturation, annealing, and extension. (Each of these steps will be explained in detail later on in the paper). After completion of these three steps, the experiment cycles back and the cycle begins again at the annealing step for 39 cycles (Life Technologies).
During the qPCR experiment, the amplified produced is measured at the end of each cycle. This is the key difference between quantitative PCR and regular PCR, as regular PCR measures the amount of amplified product produced only at the end of the reaction. Due to the product being measured at the end of each cycle, researches can use qPCR to determine the initial quantity of starting cDNA, corresponding to the region being amplified(Life Technologies).
The reaction solution includes: SYBR green (Quanta Biosciences, catalog number 95056-100), forward primer, reverse primer, cDNA, nuclease free water. SYBR green is an interlacing fluorescent dye that binds to the minor grove of any double stranded DNA, the dye only omits fluoresce when bound to a double stranded molecule. The forward and reverse primers bind to the region on the cDNA they are specific for and the nuclease free water is added to increase the volume of the solution to 10µL. (Life Technologies).
When the initial reaction solution is prepared, the cDNA is double stranded, thus the SYBR green dye binds to the minor groove and fluorescence occurs. During the denaturation step, the reaction is heated to 95. This high temperature results in the dissociation of the double stranded cDNA. Now that the cDNA is single stranded, the SYBR green will not bind to it, and the primers have room to bind and amplify. The next step in the reaction is the annealing step, the temperature of this step is important because the correct temperature is needed to allow the primers to bind to the cDNA. During the extension step, primer extension occurs at rates of up to 100 base pairs per second. The reaction then cycles back to the annealing step, and the cycle repeats 39 times. (Life Technologies).
As the extension occurs, the amplified product is double stranded, thus SYBR green binds to it. As more product is accumulated, more SYBR green is able to bind, thus more fluorescence is expressed. After each cycle, the qPCR machine measures the amount of product by measuring the amount of fluorescent signal. The baseline of the qPCR reaction is the fluorescence signal that occurs during the initial stages of the reaction. The cycle number corresponding to fluorescence that increases past the baseline threshold, is referred to as the threshold cycle (Ct). The Ct is inversely related to the initial quantity of cDNA and therefore the Ct number is used for analysis. The Ct numbers generated during the reaction are analyzed by the ∆∆Ct method. This method is used for relative gene expression, aka- finding the expression of a gene relative to a control group (Life Technologies).
As previously stated, the region that is amplified depends on where the primers bind. Therefore, the accuracy of the binding results directly depends on how specific the primers are. Two ways to ensure primer specify are blasting primer sequence to ensure it matches to correct gene, and generating melting curves of primers.
Also, as previously stated the SYBR green dye binds to any double stranded molecule. Therefore, any non-specific region that is amplified or any primer-dimers that are formed will also give a fluorescent signal. A way to check that this is not happening in a reaction is to look at a melting curve of the reaction after the reaction is complete.
Melting curves are generated once the reactions 39 cycles are complete. The reaction is heated back up to 95, which dissociates all of the amplified product. Once the produced dissociates, it is single stranded and thus SYBR green no longer omits a fluorescent signal. The qPCR machine monitors this change in fluorescent signal and this data is what is used to generate the melting curve. If the SYBR green stops omitting a fluorescent signal at the same temperature, then the reaction amplified only one product. This would be shown in the melting curve as one single, sharp peak. Thus melting curves are a way to check primer specificity and a way to double check what the SYBR green is binding to.(Life Technologies).