Improved Cardiac Characteristics In Tibetan Antelope During Adaptation To High Altitude And The Gene Clone And Expression Of CaMKⅡδ, ANP And BNP

Background and Objective: The Qinghai-Tibetan Plateau is the highest and biggest plateau in the world, with an area of 2.5 million square kilometers and an average altitude of over 4000 meters. The flora and fauna on the plateau are constantly challenged by a harsh environment characterized by low atmospheric oxygen, low temperature, and high solar radiation, all challenging a reproductive success. The high altitude hypoxia is the most important ecological factor restricting viability of residents of plateau; humans as well as animals. Native animals on the Qinghai-Tibetan Plateau have survived there for thousands of years and have developed their own mechanisms of adaptation to harsh environmental stress during their long evolutionary history. The elucidation of the evolutionary adaptation of native animals should shed light on the pathological responses non- adapted lowlanders who experience upon short and prolonged exposure to high altitude including acute and chronic mountains sickness.Previous studies about the native animals such as Yak, Pika and Snow Pig indicated that they are some physiological adaptive characteristics, which include the blunted hypoxic ventilatory response, lower hemoglobin concentration, lack of muscularization of pulmonary arteries, high affinity of hemoglobin for O2, small erythrocytes and no mast cell in lung capillaries.The heart is responsible for pumping blood throughout the blood vessels by repeated, rhythmic contractions. Calcium (Ca2+) plays an important role in excitation-contraction (E-C) coupling of cardiomyocyte, and CaMKII is a ubiquitous mediator in calcium signaling and modulates calcium handling and physiological processes in cardiomyocytes such as excitation-contraction coupling, gene transcription and apoptosis. ANP and BNP are cardiac hormones and markers of cardiac hypertrophy and congestive heart failure, and the secretion of ANP is regulated by intracellular Ca2+ via the ryanodine receptor and CaMKII.Tibetan Antelope (Pantholops hodgsonii), also called chiru, is a large mammal that is completely endemic to the Tibetan Plateau. The chiru lives at elevations of 3,700–5,500 m,an environment where it is very cold, has high UV exposure and where the atmosphere is short of oxygen. Despite living at this high altitude, with its shortage of oxygen, the chiru can still run at a speed of 80 km/h, with maximum speeds reported to be 120 km/h. Not only can the chiru run at high speed, but they can run for hours at this spectacular speed in an extremely hypoxic environment. The fact that they can run at such high speed and have stamina indicates that they are supremely adapted for life at high altitude.To investigate the mechanisms of the morphological, physiological and biochemical adaptation to high altitudes on the heart of Tibetan antelope, we compared the potential difference in left ventricular contractility, morphology of heart, and the expression of cardiac related genes(CaMKⅡδ, ANP, BNP) between Tibetan antelope and Tibetan sheep.Methods: In accordance with approval from the State Forestry Administration (SFA) and the Guide for the Care and Use of Laboratory Animals by the Ministry of Science and Technology of the People’s Republic of China, 9 male Tibetan antelope and 10 male Tibetan sheep were captured from Kekexili Natural Reservation (altitude 4300 m) and transported to Gurmud (altitude 2800 m), where the study was conducted immediately after the arrival of the animals. Animals were weighed and then anesthetized with an intraperitoneal injection of xylazole hydrochloride (1-5 mg/kg body weight) and 500 units of heparin. Cardiac catheterization was performed, and the catheter was inserted into the left ventricle via the left carotid artery. After animals recovered awake, the data were collected. After 20 min stabilization, the animals were exposed to hypoxic-gas with a venting system of one-way flap valve to mask ventilation, in which the fraction of inspired O2 (FiO2) ranged from 14.6% to 12.5%. Data were obtained by catheterization using a Biopac MP100 series and analyzed using the Acqknowledge 3.5.2 software. Data included heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and maximal rate of left ventricular (LV) pressure rise in systole (+dp/dt), and maximum rate of LV pressure fall in diastole (-dp/dt). The hearts from 5 Tibetan antelope and 5 Tibetan sheep were excised for morphologic examination and tissue collection.Cardiac and right ventricular (RV) hypertrophy was assessed by the ratio of the heart to body weight (HW/BW) and the RV to LV plus interventricular septum, RV/(LV +IVS). The Ultra-structural changes of the cardiac muscle were also observed by Electro microscope.The expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and calcium/calmodulin- dependent protein kinase II delta (CaMKⅡδ) was determined by real time PCR.Results:(1) Morphologic results : The HW/BW of Tibetan antelope (8.38±0.30%) was significantly greater than that of Tibetan sheep (4.84±0.52%) (P < 0.001), and the RV/(LV+IVS) was the same in both animals (Tibetan antelope: 0.34±0.02%, Tibetan sheep: 0.36±0.05%. Ultra-structural of the cardiac muscle indicate there were abundant mitochondria in the cardiac of the antelope when compared to the sheep.(2) Hemodynamic measurements: At baseline, the HR, SBP and DBP were 26.33±6.15 beat/min, 97.75±9.56 mmHg, and 69.53±7.55 mmHg in Tibetan antelope, respectively, while they were 34.20±6.57 beat/min, 130.06±17.79 mmHg, and 91.70±13.58 mmHg in Tibetan sheep, respectively. These parameters were significantly lower in Tibetan antelope than in Tibetan sheep (P < 0.05). However, the value of±dp/dt showed no significant difference between Tibetan antelope and Tibetan sheep (P > 0.05), which were +633.87±159.49 mmHg/s and -396.93±166.68 mmHg/s in the former, and +564.76±229.03 mmHg/s and -259.82±124.21 mmHg/s in the latter. After exposure to 14.6% and 12.5% hypoxic-gas (which were used to simulate altitudes of 5300 m and 6300 m, respectively), the +dp/dt of Tibetan antelope increased significantly while that of Tibetan sheep decreased. The values of SBP, DBP and -dp/dt in Tibetan antelope were similar to the values at the baseline. Nevertheless, the values of SBP and DBP decreased significantly in Tibetan sheep although the -dp/dt did not change significantly . The values at FiO2 21.1% were regarded as the baseline (100%). After exposure to 14.6% and 12.5% hypoxic-gas, the +dp/dt of Tibetan antelope was significantly increased to 145.1% and 148.1%, respectively, while that of the Tibetan sheep was decreased to 68.4% to 70.5%, respectively .(3) Clone and expression of CaMKⅡδ, ANP, BNP and GAPDH: The partial sequences for these genes have been accepted by GeneBank as follows: CaMKII (Tibetan antelope access no: HQ230328, Tibetan sheep access no: HQ449191), ANP (Tibetan antelope access no: HQ449188, Tibetan sheep access no: HQ449190), BNP (Tibetan antelope access no: HQ449187, Tibetan sheep access no: HQ449189), and GAPDH (Tibetan antelope access no: HQ625519, Tibetan sheep access no: HQ625518). The expression of CaMKIIδand ANP was significantly increased in Tibetan antelope compared to Tibetan sheep, but BNP expression showed no significant difference between Tibetan antelope and Tibetan sheepConclusions:(1) Tibetan antelope have myocardial hypertrophy, Physiological hypertrophy is an adaptive response that maintains cardiac function by increasing the size of cardiomyocytes, thus enhancing hemodynamic loads under physiological conditions We therefore propose that the heart of Tibetan antelope may adapt to high altitudes by physiological hypertrophy.(2) Tibetan antelope adapts to high altitude hypoxia by lowering HR, SBP and heart contractility, which thus keeps oxygen consumption at lower level,but under conditions of hypoxic stress, the increased +dp/dt of the left ventricle of Tibetan antelope contribute to oxygen delivery to tissues.(3) Ultra-structural of the cardiac muscle indicate there were abundant mitochondria in the cardiac of the tibetan antelope when compared to the tibetan sheep which can enhance the ability to produce energy in heart. (4) The partial sequences of CaMKⅡδ, ANP, BNP and GAPDH was successful cloned and accepted by GeneBank .(5) The increased expression of CaMKIIδmRNA was accompanied by increased ANP mRNA level, which may contribute to cardiac physiological hypertrophy to keep calcium homeostasis and ventricular contraction in Tibetan antelope. The unchanged expression of BNP implies that there is no cardiac dysfunction in the heart of Tibetan antelope.In conclusion, the heart of Tibetan antelope is well adapted to high altitude hypoxia characterized by increased heart weight, increased cardiac contractility under stress conditions, and increased expression of key genes regulating cardiac contractility and cardiac hypertrophy .